Synthetic apelin mimetics for the treatment of heart failure

ABSTRACT

The invention provides a synthetic polypeptide of Formula I′: 
     
       
         
         
             
             
         
       
         
         
           
             or an amide, an ester or a salt thereof, wherein X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12 and X13 are defined herein. The polypeptides are agonist of the APJ receptor. The invention also relates to a method for manufacturing the polypeptides of the invention, and its therapeutic uses such as treatment or prevention of acute decompensated heart failure (ADHF), chronic heart failure, pulmonary hypertension, atrial fibrillation, Brugada syndrome, ventricular tachycardia, atherosclerosis, hypertension, restenosis, ischemic cardiovascular diseases, cardiomyopathy, cardiac fibrosis, arrhythmia, water retention, diabetes (including gestational diabetes), obesity, peripheral arterial disease, cerebrovascular accidents, transient ischemic attacks, traumatic brain injuries, amyotrophic lateral sclerosis, burn injuries (including sunburn) and preeclampsia. The present invention further provides a combination of pharmacologically active agents and a pharmaceutical composition.

FIELD OF THE INVENTION

The invention relates to novel compositions comprising modified peptideand polypeptide sequences designed to treat cardiovascular disease insubjects to whom they are administered, and which exhibit greaterresistance to degradation, and equivalent or greater bioactivity thantheir wild type counterparts. The invention also relates to methods ofmaking said compositions and using said compositions as pharmaceuticallyactive agents to treat cardiovascular disease.

BACKGROUND OF THE INVENTION

The incidence of heart failure in the Western world is approximately1/100 adults after 65 yrs of age. The most common pathology is a chronicdeficit in cardiac contractility and, thereby, cardiac output, i.e., theeffective volume of blood expelled by either ventricle of the heart overtime. Patients with chronic heart failure can have acute episodes ofdecompensation, i.e., failure of the heart to maintain adequate bloodcirculation, where cardiac contractility declines further. There are˜500K hospitalizations per year for “acute decompensated heart failure”(ADHF) in the USA alone.

Current therapies for ADHF include diuretics, vasodilators, andinotropes, which directly increase cardiac contractility. Currentintravenous inotropes (dobutamine, dopamine, milrinone, levosimendan)are used in the acute setting, despite their association with adverseevents such as arrhythmia and increased long-term mortality. Theseliabilities have prevented their application in chronic heart failure.Digoxin is an oral inotrope, but is limited by a narrow therapeuticindex, increased arrhythmogenic potential and contraindication in renalinsufficiency.

A therapy for heart failure that increases cardiac contractility withoutarrhythmogenic or mortality liabilities is urgently needed for ADHF, butcould also address the enormous unmet medical need in chronic heartfailure.

Apelin is the endogenous ligand for the previously orphanG-protein-coupled receptor (GPCR), APJ, also referred to as apelinreceptor, angiotension-like-1 receptor, angiotension II-like-1 receptor,and the like. The apelin/APJ pathway is widely expressed in thecardiovascular system and apelin has shown major beneficialcardiovascular effects in preclinical models. Acute apelinadministration in humans causes peripheral and coronary vasodilatationand increases cardiac output (Circulation. 2010; 121:1818-1827). As aresult, APJ agonism is emerging as an important therapeutic target forpatients with heart failure. Activation of the apelin receptor APJ isthought to increase cardiac contractility and provide cardioprotection,without the liabilities of current therapies. However, the nativeapelins exhibit a very short half life and duration of action in vivo.

It is therefore desirable to identify peptides and polypeptides thatmimic the function of apelin, but have increased half-life anddemonstrate equivalent or greater bioactivity than the naturallyoccurring apelin. Furthermore, it is desirable to identify apelin analogpeptides and polypeptides which exhibit increased conformationalconstraints, i.e., the ability to achieve and maintain an activeconformational state such that the peptides and polypeptides caninteract with their receptors and/or other pathway targets without theneed for additional folded or repositioning. There is a need for use ofsuch peptide and polypeptide analogs, compositions comprising saidanalogs, and methods of making and using said compositions aspharmaceutically active agents to treat diseases, such as cardiovasculardiseases.

SUMMARY OF THE INVENTION

The aim of the present invention is to provide novel peptides andpolypeptides which are useful as APJ agonists, and which also possess atleast one of the following improvements over wild type apelin and otherknown apelin analogs: increased half-life; greater immunity todegradation upon administration and/or upon solubilization; andincreased conformational constraints, all while exhibiting the same orgreater biological activity as wild type apelin. The peptides andpolypeptides of this invention are thus particularly useful for thetreatment or prevention of cardiovascular diseases such as heartfailure, disorders and conditions associated with heart failure, anddisorders and conditions responsive to the activation of APJ receptoractivity.

In one embodiment, the peptides and polypeptides of the invention areparticularly useful for the treatment or prevention of a disorder orcondition associated with heart failure, or a disorder responsive to theactivation (or agonism) of the APJ receptor activity. In anotherembodiment, the peptides and polypeptides of the invention are useful inthe treatment of acute decompensated heart failure (ADHF), chronic heartfailure, pulmonary hypertension, atrial fibrillation, Brugada syndrome,ventricular tachycardia, atherosclerosis, hypertension, restenosis,ischemic cardiovascular diseases, cardiomyopathy, cardiac fibrosis,arrhythmia, water retention, diabetes (including gestational diabetes),obesity, peripheral arterial disease, cerebrovascular accidents,transient ischemic attacks, traumatic brain injuries, amyotrophiclateral sclerosis, burn injuries (including sunburn) and preeclampsia.

The invention pertains to the peptides and polypeptides, pharmaceuticalcompositions, and methods of manufacture and use thereof, as describedherein. Examples of peptides and polypeptides of the invention includethe peptides and polypeptides according to any one of Formulae I to IX,or an amide, an ester or a salt thereof, as well as any peptides orpolypeptides specifically listed herein, including but not limited tothe experimental examples.

The invention therefore provides a peptide or a polypeptide formula(I′):

wherein:X1 is the N-terminus of the polypeptide and is either absent or isselected from pE, R, Isn, Q, A, K, and 5-amino-valeric acid;

X2 is R, A, r, N-Me-R, K, H, hF, hK, F, E or Orn;

X3 is P, A, a, p, 4-PhP, K, D, pipecolic acid, or cysteine wherein theside chain of cysteine forms a disulfide bond with the side chain of thecysteine at the X7 position;X4 is R, A, r, N-Me-R, F, E or cysteine wherein the side chain ofcysteine forms a disulfide bond with the side chain of the cysteine atthe X7 position;

X5 is L, Cha, A, D-L, N-Me-L, K, D, 4-PhF or F;

X6 and X12 are independently a natural or unnatural amino acid selectedfrom C, c, hC, D-hC, K, D, Orn, Dab or E wherein the side chain of X6and X12 are linked together via a covalent bond forming either amonosulfide (—S—), a disulfide (—S—S—) or an amide bond (—NHC(O)— or—C(O)—NH—) or a linkage of Formula —S—CH₂—C(═Z)—CH₂—S—; wherein Z is O,N—O—CH₂C(O)-L¹-R¹ or N—NH—CH₂C(O)-L¹-R¹ wherein R¹ is a fatty acidderivative and L¹ is a linker containing one or more amino acids and/orone or more —NH—C₂₋₆-alkylene-NH— units; or alternatively X6 is K, X13is absent and X12 is F or f wherein the C-terminus of X12 form an amidebond with the amino side chain of X6;X7 is H, h, A, N-Me-A, a, Aib, K, Nal, F, P, Dap, N, E or cysteinewherein the side chain of the cysteine forms a disulfide bond with theside chain of the cysteine at position X3 or with the side chain of thecysteine at position X4;X8 is K, k, F, f, A, hF, N-Me-R, E or 4-amino-Isn;

X9 is G, N-Me-G, A, D, L, R or Aib;

X10 is P, A, p, 4-PhP or pipecolic acid,

X11 is M, D-Nle, Nle, N-Me-Nle, M(O), A, F, Y, L, K, 3-PyA or Cha; and

X13 is the C-terminus and is absent or is selected from F, f, N-Me-F,Nal, D-Nal, 3-Br—F, (S)-β-3-F, I, A, a, K, Dap, H and E;wherein:

Nle is L-norleucine; D-hC is D-homocysteine hC is L-homocysteine; hF isL-homophenylalanine; hK is L-lysine; Nal is L-naphathaline;

Orn is ornithine;Aib is α-aminoisobutyric acid;Dab is (S)-diaminobutyric acid;Dap is (S)-2,3-diaminopropionic acid;M(O) is methionine sulfone;Cha is (S)-β-cyclohexylalanine;4-amino-Isn is 4-aminopiperidine-4-carboxylic acid;Isn is isonipecotinoyl;pE is L-pyroglutamic acid;3-PyA is 3-(3-pyridyl)-L-alanine;

4-PhF is 4-Phenyl-L-phenylalanine;

wherein the N-terminus and the C-terminus optionally form a ringtogether with 1, 2, 3 or 4 glycine amino acids; andwherein the amino group in the side chain of K, Orn, Dab, Dap, hK or4-amino-Isn is optionally linked to a lipophilic group via an amidebond;or an amide, an ester or a salt of the polypeptide; or a polypeptidesubstantially equivalent thereto.

As further explained herein, the art-recognized three letter or oneletter abbreviations are used to represent amino acid residues thatconstitute the peptides and polypeptides of the invention. Except whenpreceded with “D,” the amino acid is an L-amino acid. When the oneletter abbreviation is a capital letter, it refers to the L-amino acid.When the one letter abbreviation is a lower case letter, it refers tothe D-amino acid.

Any of the above-listed amino acid residues of Formula I′, or itsrelated formulae described herein, e.g., Formulae I, II to IX, may besubstituted in a conservative fashion, provided the peptide orpolypeptide of the invention still retains functional activity andstructural properties (e.g., half-life extension, protection fromdegradation, conformational constraint). Principle and examples ofpermissible conservative amino acid substitutions are further explainedherein.

The polypeptides of the invention, by activating APJ receptor, haveutility in the treatment of

acute decompensated heart failure (ADHF), chronic heart failure,pulmonary hypertension, atrial fibrillation, Brugada syndrome,ventricular tachycardia, atherosclerosis, hypertension, restenosis,ischemic cardiovascular diseases, cardiomyopathy, cardiac fibrosis,arrhythmia, water retention, diabetes (including gestational diabetes),obesity, peripheral arterial disease, cerebrovascular accidents,transient ischemic attacks, traumatic brain injuries, amyotrophiclateral sclerosis, burn injuries (including sunburn) and preeclampsia.

In a preferred embodiment the polypeptides of the invention are usefulin the treatment of acute decompensated heart failure (ADHF).

In another embodiment, the invention pertains to a method for treatingdisorder or disease responsive to the activation of the APJ receptor, ina subject in need of such treatment, comprising: administering to thesubject an effective amount of a polypeptide according to anyone ofFormulae I to IX, or an amide, an ester of a salt thereof, such that thedisorder or disease responsive to the activation of the APJ receptor inthe subject is treated.

In yet another embodiment, the invention pertains to pharmaceuticalcompositions, comprising a polypeptide according to anyone of Formulae Ito IX, or an amide, an ester or salt thereof, and one or morepharmaceutically acceptable carriers.

In still another embodiment, the invention pertains to combinationsincluding, a polypeptide according to anyone of Formulae I to IX, or anamide, an ester or a salt thereof, and pharmaceutical combinations ofone or more therapeutically active agents.

In another embodiment, the invention pertains to a method for activationof the APJ receptor in a subject in need thereof, comprising:administering to the subject a therapeutically effective amount of apolypeptide according to anyone of Formulae I to IX, or an amide, anester or a salt thereof.

DETAILED DESCRIPTION OF THE INVENTION

For purposes of interpreting this specification, the followingdefinitions will apply unless specified otherwise and wheneverappropriate, terms used in the singular will also include the plural andvice versa.

As used herein, “disorders or diseases responsive to the modulation ofthe APJ receptor,” “disorders and conditions responsive to themodulation of the APJ,” “disorders and conditions responsive to themodulation of APJ receptor activity,” “disorders responsive to theactivation (or agonism) of the APJ receptor activity” and like termsinclude acute decompensated heart failure (ADHF), chronic heart failure,pulmonary hypertension, atrial fibrillation, Brugada syndrome,ventricular tachycardia, atherosclerosis, hypertension, restenosis,ischemic cardiovascular diseases, cardiomyopathy, cardiac fibrosis,arrhythmia, water retention, diabetes (including gestational diabetes),obesity, peripheral arterial disease, cerebrovascular accidents,transient ischemic attacks, traumatic brain injuries, amyotrophiclateral sclerosis, burn injuries (including sunburn) and preeclampsia.

As used herein, “Activation of APJ receptor activity,” or “Activation ofthe APJ receptor,” refers to an increase in the APJ receptor activity.The activation of the APJ receptor activity is also referred to as“agonism” of the APJ receptor, e.g., by administration of the peptidesand polypeptides of the invention.

As used herein, the terms “polypeptide” and “peptide” are usedinterchangeably to refer to two or more amino acids linked together.Except for the abbreviations for the uncommon or unatural amino acidsset forth in Table 1 below, the art-recognized three letter or oneletter abbreviations are used to represent amino acid residues thatconstitute the peptides and polypeptides of the invention. Except whenpreceded with “D”, the amino acid is an L-amino acid. When the oneletter abbreviation is a capital letter, it refers to the D-amino acid.When the one letter abbreviation is a lower case letter, it refers tothe L-amino acid. Groups or strings or amino acid abbreviations are usedto represent peptides. Peptides are indicated with the N-terminus on theleft and the sequence is written from the N-terminus to the C-terminus.

Peptides of the invention contain non-natural amino acids (i.e.,compounds that do not occur in nature) and other amino acid analogs asare known in the art may alternatively be employed.

Certain non-natural amino acids can be introduced by the technologydescribed in Deiters et al., J Am Chem Soc 125:11782-11783, 2003; Wangand Schultz, Science 301:964-967, 2003; Wang et al., Science292:498-500, 2001; Zhang et al., Science 303:371-373, 2004 or in U.S.Pat. No. 7,083,970. Briefly, some of these expression systems involvesite-directed mutagenesis to introduce a nonsense codon, such as anamber TAG, into the open reading frame encoding a polypeptide of theinvention. Such expression vectors are then introduced into a host thatcan utilize a tRNA specific for the introduced nonsense codon andcharged with the non-natural amino acid of choice. Particularnon-natural amino acids that are beneficial for purpose of conjugatingmoieties to the polypeptides of the invention include those withacetylene and azido side chains.

One or more of the natural or un-natural amino acids in a peptide of theinvention may be modified, for example, by the addition of a chemicalentity such as a carbohydrate group, a phosphate group, a farnesylgroup, an isofarnesyl group, a fatty acid group (C_(q)H_(q+1)C(O)₂Hwherein q is 3 to 20), a linker for conjugation, functionalization, orother modification, etc. Said modifications may be done in asite-specific or non-site-specific manner. In a preferred embodiment,the modifications of the peptide lead to a more stable peptide (e.g.,one exhibiting greater half-life in vivo). These modifications mayinclude the incorporation of additional D-amino acids, etc. None of themodifications should substantially interfere with the desired biologicalactivity of the peptide, but such modifications may confer desirableproperties, e.g., enhanced biological activity, on the peptide.

Said modifications enhance the biological properties of the proteins ofthe invention relative to the wild-type proteins, as well as, in somecases, serving as points of attachment for, e.g., labels and proteinhalf-life extension agents, and for purposes of affixing said variantsto the surface of a solid support.

In certain embodiments, such modifications, e.g., site-specificmodifications, are used to attach conjugates, e.g., PEG groups topolypeptides, and/or peptides of the invention, for purposes of, e.g.,extending half-life or otherwise improving the biological properties ofsaid polypeptides, and/or peptides. Said techniques are describedfurther herein.

In other embodiments, such modifications, e.g., site-specificmodifications, are used to attach other polymers and small molecules andrecombinant protein sequences that extend half-life of the polypeptideof the invention. One such embodiment includes the attachment of fattyacids or specific albumin binding compounds to polypeptides, and/orpeptides. In other embodiments, the modifications are made at aparticular amino acid type and may be attached at one or more sites onthe polypeptides.

In other embodiments, such modifications, e.g., site-specificmodifications, are used as means of attachment for the production ofwild-type and/or variant multimers, e.g., dimers (homodimers orheterodimers) or trimers or tetramers. These multimeric proteinmolecules may additionally have groups such as PEG, sugars, and/orPEG-cholesterol conjugates attached or be fused either amino-terminallyor carboxy-terminally to other proteins such as Fc, Human Serum Albumin(HSA), etc.

In other embodiments, such site-specific modifications are used toproduce proteins, polypeptides and/or peptides wherein the position ofthe site-specifically incorporated pyrrolysine or pyrrolysine analogueor non-naturally occurring amino acids (para-acetyl-Phe, para-azido-Phe)allows for controlled orientation and attachment of such proteins,polypeptides and/or peptides onto a surface of a solid support or tohave groups such as PEG, sugars and/or PEG-cholesterol conjugatesattached.

In other embodiments, such site-specific modifications are used tosite-specifically cross-link proteins, polypeptides and/or peptidesthereby forming hetero-oligomers including, but not limited to,heterodimers and heterotrimers. In other embodiments, such site-specificmodifications are used to site-specifically cross-link proteins,polypeptides and/or peptides thereby forming protein-protein conjugates,protein-polypeptide conjugates, protein-peptide conjugates,polypeptide-polypeptide conjugates, polypeptide-peptide conjugates orpeptide-peptide conjugates. In other embodiments, a site specificmodification may include a branching point to allow more than one typeof molecule to be attached at a single site of a protein, polypeptide orpeptide.

In other embodiments, the modifications listed herein can be done in anon-site-specific manner and result in protein-protein conjugates,protein-polypeptide conjugates, protein-peptide conjugates,polypeptide-polypeptide conjugates, polypeptide-peptide conjugates orpeptide-peptide conjugates of the invention.

In some embodiments, the present invention provides complexes whichcomprise at least one peptide or polypeptide of anyone of Formulae I-IXbound to an antibody, such as an antibody why specifically binds apeptide or polypeptide as disclosed herein.

One of ordinary skill in the art will appreciate that various amino acidsubstitutions, e.g, conservative amino acid substitutions, may be madein the sequence of any of the polypeptides described herein, withoutnecessarily decreasing its activity. As used herein, “amino acidcommonly used as a substitute thereof” includes conservativesubstitutions (i.e., substitutions with amino acids of comparablechemical characteristics). For the purposes of conservativesubstitution, the non-polar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, glycine, proline, phenylalanine, tryptophanand methionine. The polar (hydrophilic), neutral amino acids includeserine, threonine, cysteine, tyrosine, asparagine, and glutamine. Thepositively charged (basic) amino acids include arginine, lysine andhistidine. The negatively charged (acidic) amino acids include asparticacid and glutamic acid. Examples of amino acid substitutions includesubstituting an L-amino acid for its corresponding D-amino acid,substituting cysteine for homocysteine or other non natural amino acidshaving a thiol-containing side chain, substituting a lysine forhomolysine, diaminobutyric acid, diaminopropionic acid, ornithine orother non natural amino acids having an amino containing side chain, orsubstituting an alanine for norvaline or the like.

The term “amino acid,” as used herein, refers to naturally occurringamino acids, unnatural amino acids, amino acid analogues and amino acidmimetics that function in a manner similar to the naturally occurringamino acids, all in their D and L stereoisomers if their structureallows such stereoisomeric forms. Amino acids are referred to herein byeither their name, their commonly known three letter symbols or by theone-letter symbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission.

The term “naturally occurring” refers to materials which are found innature and are not manipulated by man. Similarly, “non-naturallyoccurring,” “un-natural,” and the like, as used herein, refers to amaterial that is not found in nature or that has been structurallymodified or synthesized by man. When used in connection with aminoacids, the term “naturally occurring” refers to the 20 conventionalamino acids (i.e., alanine (A or Ala), cysteine (C or Cys), asparticacid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe),glycine (G or Gly), histidine (H or His), isoleucine (I or Ile), lysine(K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N orAsn), proline (P or Pro), glutamine (Q or Gln), arginine (R or Arg),serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan(W or Trp), and tyrosine (Y or Tyr)).

The terms “non-natural amino acid” and “unnatural amino acid,” as usedherein, are interchangeably intended to represent amino acid structuresthat cannot be generated biosynthetically in any organism usingunmodified or modified genes from any organism, whether the same ordifferent. The terms refer to an amino acid residue that is not presentin the naturally occurring (wild-type) apelin protein sequence or thesequences of the present invention. These include, but are not limitedto, modified amino acids and/or amino acid analogues that are not one ofthe 20 naturally occurring amino acids, selenocysteine, pyrrolysine(Pyl), or pyrroline-carboxy-lysine (Pcl, e.g., as described in PCTpatent publication WO2010/48582). Such non-natural amino acid residuescan be introduced by substitution of naturally occurring amino acids,and/or by insertion of non-natural amino acids into the naturallyoccurring (wild-type) Apelin protein sequence or the sequences of theinvention. The non-natural amino acid residue also can be incorporatedsuch that a desired functionality is imparted to the apelin molecule,for example, the ability to link a functional moiety (e.g., PEG). Whenused in connection with amino acids, the symbol “U” shall mean“non-natural amino acid” and “unnatural amino acid,” as used herein.

In addition, it is understood that such “unnatural amino acids” requirea modified tRNA and a modified tRNA synthetase (RS) for incorporationinto a protein. These “selected” orthogonal tRNA/RS pairs are generatedby a selection process as developed by Schultz et al. or by random ortargeted mutation. As way of example, pyrroline-carboxy-lysine is a“natural amino acid” as it is generated biosynthetically by genestransferred from one organism into the host cells and as it isincorporated into proteins by using natural tRNA and tRNA synthetasegenes, while p-aminophenylalanine (See, Generation of a bacterium with a21 amino acid genetic code, Mehl R A, Anderson J C, Santoro S W, Wang L,Martin A B, King D S, Horn D M, Schultz P G. J Am Chem Soc. 2003 Jan.29; 125(4):935-9) is an “unnatural amino acid” because, althoughgenerated biosynthetically, it is incorporated into proteins by a“selected” orthogonal tRNA/tRNA synthetase pair.

Modified encoded amino acids include, but are not limited to,hydroxyproline, carboxyglutamate, O-phosphoserine, azetidinecarboxylicacid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine,aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid,6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid,3-aminoisobutyric acid, 2-aminopimelic acid, tertiary-butylglycine,2,4-diaminoisobutyric acid, desmosine, 2,2′-diaminopimelic acid,2,3-diaminoproprionic acid, N-ethylglycine, N-methylglycine,N-ethylasparagine, homoproline, hydroxylysine, allo-hydroxylysine,3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine,N-methylalanine, N-methylglycine, N-methylisoleucine,N-methylpentylglycine, N-methylvaline, naphthalanine, norvaline,norleucine, ornithine, pentylglycine, pipecolic acid and thioproline.The term “amino acid” also includes naturally occurring amino acids thatare metabolites in certain organisms but are not encoded by the geneticcode for incorporation into proteins. Such amino acids include, but arenot limited to, ornithine, D-ornithine, and D-arginine.

The term “amino acid analogue,” as used herein, refers to compounds thathave the same basic chemical structure as a naturally occurring aminoacid, by way of example only, an α-carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group. Amino acid analoguesinclude the natural and unnatural amino acids which are chemicallyblocked, reversibly or irreversibly, or their C-terminal carboxy group,their N-terminal amino group and/or their side-chain functional groupsare chemically modified. Such analogues include, but are not limited to,methionine sulfoxide, methionine sulfone, S-(carboxymethyl)-cysteine,S-(carboxymethyl)-cysteine sulfoxide, S-(carboxymethyl)-cysteinesulfone, aspartic acid-(beta-methyl ester), N-ethylglycine, alaninecarboxamide, homoserine, norleucine, and methionine methyl sulfonium.

TABLE 1 Un-natural or Non-natural Amino Acids as described in theinvention: Symbol Name Structure Aib α-Aminoisobutyric acid

M(O) Methionine sulfone

1-Nal 1-Naphthalanine

2-Nal 2-Naphthalanine

Cha β-Cyclohexylalanine

Dab Diaminobutyric acid

Dap 2,3-Diamino propionic acid

hC Homocysteine

hF Homophenylalanine

hK Homolysine

Nle Norleucine

Orn Ornithine

β-3-F β-3-phenylalanine

4- amino- Isn 4-Aminopiperidine- 4-carboxylic acid (4 amino group formthe peptidic bond)

Isn Isonipecotinoic acid

pE Pyroglutamic acid

4-PhP 4-Phenylproline

Pipecolinic acid

5-Aminovaleric acid

O2Oc 8-Amino-3,6-dioxa- octanoic acid

3-PyA 3-(3-pyridyl)- alanine

4-PhF 4-phenyl- phenylalanine

Nal refers both to 1-Naphthalanine and 2-Naphthalanine, preferably2-naphthalanine. 4-Phenylproline refers to both cis and trans4-Phenylproline, preferably trans-4-phenylproline

As used herein the term “amide” refers to an amide derivative of thecarboxylic acid group at the C-terminus (e.g. —C(O)NH₂, —C(O)NH—C₁₋₆alkyl, —C(O)NH—C1-2alkylphenyl, —C(O)NH—NHBn or —C(O)N(C₁₋₆ alkyl)₂).

The term “amide” also refer to derivative of the amino group at theN-terminus (e.g. —NHC(O)C₁₋₁₆alkyl, —NHC(O)(CH₂)_(n)Ph (n is an integerof 1 to 6), —NHC(O)(CH₂)₂CO₂H, 4-Cl-Ph-(CH₂)₃C(O)NH—,C₁₁H₂₃C(O)NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)—NH—,C₁₃H₂₇C(O)NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)—NH—;C₁₆H₂₇C(O)NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)NH—, Ph-CH₂CH₂NHC(O)—NH— orCH₃(OCH₂CH₂)_(m)C(O)NH— (m is an integer of 1 to 12).

As used herein, the term “ester” refers to an ester derivative of thecarboxylic acid group at the C-terminus (e.g —COOR) form wherein R ofthe ester refers to C₁₋₆ alkyl groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, etc., C₃₋₈ cycloalkyl groups such as cyclopentyl,cyclohexyl, etc., C₆₋₁₀ aryl groups such as phenyl, α-naphthyl, etc.,C₆₋₁₀ aryl-C₁₋₆ alkyl groups, for example phenyl-C₁₋₂ alkyl groups suchas benzyl, phenethyl, benzhydryl, etc., and α-naphthyl-C₁₋₂ alkyl groupssuch as α-naphthylmethyl and the like. Mention may also be made ofpivaloyloxymethyl ester and the like, which are commonly used as estersfor oral administration. When the polypeptides of the invention possessadditional carboxyl or carboxylate groups in positions other than the Cterminus, those polypeptides in which such groups are amidated oresterified also fall under the category of the polypeptide of theinvention. In such cases, the esters may for example be the same kindsof esters as the C-terminal esters mentioned above.

The term “APJ” (also referred to as “apelin receptor,”“angiotension-like-1 receptor,” “angiotension II-like-1 receptor,” andthe like) indicates a 380 residue, 7 transmembrane domain, Gi coupledreceptor whose gene is localized on the long arm of chromosome 11 inhumans (NCBI Reference Sequence: NP_(—)005152.1, and encoded by NCBIReference Sequence: NM_(—)005161). APJ was first cloned in 1993 fromgenomic human DNA using degenerate oligonucleotide primers (O'Dowd etal. Gene, 136:355-60, 1993) and shares significant homology withangiotensin II receptor type 1. Despite this homology however,angiotensin II does not bind APJ. Although orphan for many years, theendogenous ligand has been isolated and named apelin (Tatemoto et al.,Biochem Biophys Res Commun 251, 471-6 (1998)).

The term “apelin,” indicates a 77 residue preprotein (NCBI ReferenceSequence: NP_(—)0059109.3, and encoded by NCBI Reference Sequence:NM_(—)017413.3), which gets processed into biologically active forms ofapelin peptides, such as apelin-36, apelin-17, apelin-16, apelin-13,apelin-12. The full length mature peptide, referred to as “apelin-36,”comprises 36 amino acids, but the most potent isoform is thepyroglutamated form of a 13mer of apelin (apelin-13), referred to as“Pyr-1-apelin-13 or Pyr¹-apelin-13” Different apelin forms aredescribed, for instance, in U.S. Pat. No. 6,492,324B1.

Polypeptides of the Invention:

Various embodiments of the invention are described herein. It will berecognized that features specified in each embodiment may be combinedwith other specified features to provide further embodiments.

In embodiment 1, the invention therefore provides a peptide or apolypeptide formula (I′):

wherein:X1 is the N-terminus of the polypeptide and is either absent or isselected from pE, R, Isn, Q, A, K, and 5-amino-valeric acid;

X2 is R, A, r, N-Me-R, K, H, hF, hK, F, E or Orn;

X3 is P, A, a, p, 4-PhP, K, D, pipecolic acid, or cysteine wherein theside chain of cysteine forms a disulfide bond with the side chain of thecysteine at the X7 position;X4 is R, A, r, N-Me-R, F, E or cysteine wherein the side chain ofcysteine forms a disulfide bond with the side chain of the cysteine atthe X7 position;

X5 is L, Cha, A, D-L, N-Me-L, K, D, 4-PhF or F;

X6 and X12 are independently a natural or unnatural amino acid selectedfrom C, c, hC, D-hC, K, D, Orn, Dab or E wherein the side chain of X6and X12 are linked together via a covalent bond forming either adisulfide or an amide bond;or alternatively X6 is K, X13 is absent and X12 is F or f wherein theC-terminus of X12 form an amide bond with the amino side chain of X6;X7 is H, h, A, N-Me-A, a, Aib, K, Nal, F, P, Dap, N, E or cysteinewherein the side chain of the cysteine forms a disulfide bond with theside chain of the cysteine at position X3 or with the side chain of thecysteine at position X4;X8 is K, k, F, f, A, hF, N-Me-R, E or 4-amino-Isn;

X9 is G, N-Me-G, A, D, L, R or Aib;

X10 is P, A, p, 4-PhP or pipecolic acid,

X11 is M, D-Nle, Nle, N-Me-Nle, M(O), A, F, Y, L, K, 3-PyA or Cha; and

X13 is the C-terminus and is absent or is selected from F, f, N-Me-F,Nal, D-Nal, 3-Br—F, (S)-β-3-F, I, A, a, K, Dap, H and E;wherein:

Nle is L-norleucine; D-hC is D-homocysteine hC is L-homocysteine; hF isL-homophenylalanine; hK is L-lysine; Nal is L-naphathaline;

Orn is ornithine;Aib is α-aminoisobutyric acid;Dab is (S)-diaminobutyric acid;Dap is (S)-2,3-diaminopropionic acid;M(O) is methionine sulfone;Cha is (S)-β-cyclohexylalanine;4-amino-Isn is 4-aminopiperidine-4-carboxylic acid;Isn is isonipecotinoyl;pE is L-pyroglutamic acid;3-PyA is 3-(3-pyridyl)-L-alanine;

4-PhF is 4-Phenyl-L-phenylalanine;

wherein the N-terminus and the C-terminus optionally form a ringtogether with 1, 2, 3 or 4 glycine amino acids; andwherein the amino group in the side chain of K, Orn, Dab, Dap, hK or4-amino-Isn is optionally linked to a lipophilic group via an amidebond;or an amide, an ester or a salt of the polypeptide; or a polypeptidesubstantially equivalent thereto.

In embodiment 2, the invention therefore provides a peptide or apolypeptide formula (I):

wherein:X1 is the N-terminus of the polypeptide and is either absent or isselected from pE, R, Q, A, K, 5-amino-valeric acid and Isn;

X2 is R, A, r, N-Me-R, K, H, hF, hK or Orn;

X3 is P, A, a, p, 4-PhP, pipecolic acid, or cysteine wherein the sidechain of cysteine forms a disulfide bond with the side chain of thecysteine at the X7 position;X4 is R, A, r, N-Me-R or cysteine wherein the side chain of cysteineform a disulfide bond with the side chain of the cysteine at the X7position;

X5 is L, Cha, A, D-L, N-Me-L or F;

X6 and X12 are independently a natural or unnatural amino acid selectedfrom C, c, hC, D-hC, K, D, Orn, Dab or E wherein the side chain of X6and X12 are linked together via a covalent bond;or alternatively X6 is K, X13 is absent and X12 is F or f wherein theC-terminus of X12 form an amide bond with the amino side chain of X6;X7 is H, h, A, N-Me-A, a, Aib, K, Nal, F, P, Dap, N or cysteine whereinthe side chain of the cysteine form a disulfide bond with the side chainof the cysteine at position X3 or with the side chain of the cysteine atposition X4;X8 is K, k, F, f, A, hF, N-Me-R or 4-amino-Isn;

X9 is G, N-Me-G, A or Aib;

X10 is P, A, p, 4-PhP or pipecolic acid,

X11 is M, D-Nle, Nle, N-Me-Nle, M(O), A, F, Y, L, K or Cha; and

X13 is the C-terminus and is absent or is selected from F, f, N-Me-F,Nal, D-Nal, 3-Br—F, (S)-β-3-F, I, A, a, K, Dapwherein:

Nle is L-norleucine; D-hC is D-homocysteine hC is L-homocysteine; hF isL-homophenylalanine; hK is L-lysine; Nal is L-naphathaline;

Orn is ornithine;Aib is α-aminoisobutyric acid;Dab is (S)-diaminobutyric acid;Dap is (S)-2,3-diaminopropionic acid;M(O) is methionine sulfone;Cha is (S)-β-cyclohexylalanine;4-amino-Isn is 4-Aminopiperidine-4-carboxylic acid;Isn is isonipecotinoyl;pE is L-pyroglutamic acid;wherein the N-terminus and the C-terminus optionally form a ringtogether with 1, 2, 3 or 4 glycine amino acids; andwherein the amino group in the side chain of K, Orn, Dab, Dap, hK or4-amino-Isn is optionally linked to a lipophilic group via an amidebond;or an amide, an ester or a salt of the polypeptide; or a polypeptidesubstantially equivalent thereto.

In one aspect of embodiment 1 or 2, the invention pertains to peptide orpolypeptide of Formula I or I′ wherein the amino group in the side chainof K, Orn, Dab, Dap, hK or 4-amino-Isn is optionally linked to a fattyacid via an amide bond. In a further aspect of this embodiment, thefatty acid is selected from Lauroyl, Myristoyl or Palmitoyl, whereinLauroyl is C₁₁H₂₃C(O)—, Myristoyl is C₁₃H₂₇C(O)— and Palmitoyl isC₁₅H₃₁C(O)—. Alternatively the terminal CH₃ moiety of the fatty acid(such as Lauroyl, Myristoyl or Palmitoyl) may be oxidized to itscorresponding carboxylic acid.

In one aspect of embodiment 1 or 2, the invention pertains to peptide orpolypeptide of Formula I or I′ wherein the amino group in the side chainof K, Orn, Dab, Dap, hK or 4-amino-Isn is optionally linked to alipophilic group via an amide bond, wherein the lipophilic group isselected from a fatty acid as described supra and Lauroyl(O2Oc),Myristoyl(O2Oc) and palmitoyl(O2Oc) and wherein Lauroyl(O2Oc) isC₁₁H₂₃C(O)NH—(CH₂)₂—O—(CH₂)₂—O—CH₂C(O)—; Myristoyl(O2Oc) isC₁₃H₂₇C(O)NH—(CH₂)₂—O—(CH₂)₂—O—CH₂C(O)—; palmitoyl(O2Oc) isC₁₅H₃₁C(O)NH—(CH₂)₂—O—(CH₂)₂—O—CH₂C(O)—. Alternatively the terminal CH₃moiety of the fatty acid (such as Lauroyl, Myristoyl or Palmitoyl) maybe oxidized to its corresponding carboxylic acid.

In embodiment 3, the invention pertains to a peptide or polypeptide ofFormula I or I′:

wherein:X1 is the N-terminus of the polypeptide and is either absent or isselected from pE, R, Q, A, K(palmitoyl), K(Myristoyl), K(Lauroyl),5-amino-valeric acid and Isn;

X2 is R, A, r, N-Me-R, K, H, hF, hK or Orn;

X3 is P, A, a, p, 4-PhP, pipecolic acid, or cysteine wherein the sidechain of cysteine forms a disulfide bond with the side chain of thecysteine at the X7 position;X4 is R, A, r, N-Me-R or cysteine wherein the side chain of cysteineform a disulfide bond with the side chain of the cysteine at the X7position;

X5 is L, Cha, A, D-L, N-Me-L or F;

X6 and X12 are independently a natural or unnatural amino acid selectedfrom C, c, hC, D-hC, K, D, Orn, Dab or E wherein the side chain of X6and X12 are linked together via a covalent bond;or alternatively X6 is K, X13 is absent and X12 is F or f wherein theC-terminus of X12 form an amide bond with the amino side chain of X6;X7 is H, h, A, N-Me-A, a, Aib, K(Lauroyl), K(Palmitoyl), Nal, F, P, Dap,N or cysteine wherein the side chain of the cysteine form a disulfidebond with the side chain of the cysteine at position X3 or with the sidechain of the cysteine at position X4;X8 is K, k, F, f, A, hF, N-Me-R or 4-amino-Isn;

X9 is G, N-Me-G, A or Aib;

X10 is P, A, p, 4-PhP or pipecolic acid,

X11 is M, D-Nle, Nle, N-Me-Nle, M(O), A, F, Y, L, K(Palmitoyl) or Cha;and

X13 is the C-terminus and is absent or is selected from F, f, N-Me-F,Nal, D-Nal, 3-Br—F, (S)-β-3-F, 1, A, a, K(Lauroyl), K(Myristoyl),K(palmitoyl), K(O2Oc-Palmitoyl), Dap(Palmitoyl);wherein:K(Lauroyl) is N-ε-lauroyl-L-lysine;K(palmitoyl) is N-ε-palmitoyl-L-lysine;K(Myristoyl) is N-ε-myristoyl-L-lysine;

K(O2Oc-Palmitoyl) is N-ε-(O2Oc-Palmitoyl)-L-lysine; and Palmitoyl(O2Oc)is C₁₅H₃₁C(O)NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)—; Nle is L-norleucine; D-hCis D-homocysteine hC is L-homocysteine; hF is L-homophenylalanine; hK isL-lysine; Nal is L-naphathaline;

Orn is ornithine;Aib is α-aminoisobutyric acid;Dab is (S)-diaminobutyric acid;Dap is (S)-2,3-diaminopropionic acid;M(O) is methionine sulfone;Cha is (S)-β-cyclohexylalanine;4-amino-Isn is 4-Aminopiperidine-4-carboxylic acid;Isn is isonipecotinoyl;pE is L-pyroglutamic acid;and wherein the N-terminus and the C-terminus optionally form a ringtogether with 1, 2, 3 or 4 glycine amino acids;or an amide, an ester or a salt of the polypeptide.

In embodiment 4, the invention pertains to a peptide or a polypeptideaccording to embodiment 1, 2 or 3, having Formula II:

X1 is absent, pE, R, Q or Isn;

X5 is L or Cha; X7 is H, Aib, F, K(Lauroyl) or K(palmitoyl);

X8 is K, F or 4-amino-Isn;

X9 is G or Aib; X11 is Nle or Cha;

X13 is absent or is F, f, K(Lauroyl), K(palmitoyl);X6 and X12 are independently a natural or unnatural amino acid selectedfrom C, c, hC, D-hC, K, D, Orn, Dab or E wherein the side chain of X6and X12 are linked together via a covalent bond; or alternatively X6 isK, X13 is absent and X12 is F or f wherein the C-terminus of X12 form anamide bond with the amino side chain of X6; andwherein the N-terminus and the C-terminus optionally form a ringtogether with 1, 2, 3 or 4 glycine amino acids; or an amide, an ester ora salt of the polypeptide; or a polypeptide substantially equivalentthereto.

In embodiment 5, the invention pertains to a peptide or a polypeptideaccording to embodiment 4 wherein:

X1 is absent, pE, R, Q or Isn;

X5 is L or Cha; X7 is H, Aib, F, K(Lauroyl) or K(palmitoyl);

X8 is K, F or 4-amino-Isn;

X9 is G or Aib; X11 is Nle or Cha;

X13 is absent or is F, f, K(Lauroyl), K(palmitoyl);X6 and X12 are independently a natural or unnatural amino acid selectedfrom C, K, D, Orn, Dab or E wherein the side chain of X6 and X12 arelinked together via a covalent bond; oralternatively X6 is K, X13 is absent and X12 is F or f wherein theC-terminus of X12 form an amide bond with the amino side chain of X6;andwherein the N-terminus and the C-terminus optionally form a ringtogether with 1, 2, 3 or 4 glycine amino acids; or an amide, an ester ora salt of the polypeptide; or a polypeptide substantially equivalentthereto.

In embodiment 6, a further aspect of embodiment 4, the inventionpertains to a peptide or polypetide of Formula II wherein:

X1 is absent, pE, R, Q or Isn;

X5 is L or Cha; X7 is H, Aib, F, K(Lauroyl) or K(palmitoyl);

X8 is K, F or 4-amino-Isn;

X9 is G or Aib; X11 is Nle or Cha;

X13 is absent or is F, f, K(Lauroyl), K(palmitoyl);X6 and X12 are independently a natural or unnatural amino acid selectedfrom C, c, hC, D-hC, K, D, Orn, Dab or E wherein the side chain of X6and X12 are linked together via a covalent bond; and wherein theN-terminus and the C-terminus optionally form a ring together with 1, 2,3 or 4 glycine amino acids; or an amide, an ester or a salt of thepolypeptide; or a polypeptide substantially equivalent thereto.

In yet a further aspect of anyone of the previous embodiments, morespecifically of anyone of embodiments 1 to 6, the invention pertains topeptide and polypeptide of Formula I, I′ or II wherein X6 and X12 areindependently a natural or unnatural amino acid selected from C, K, D,Orn, Dab or E wherein the side chain of X6 and X12 are linked togethervia a covalent bond; or an amide, an ester or a salt of the polypeptide;or a polypeptide substantially equivalent thereto.

In embodiment 7, the invention pertains to a peptide or polypeptide ofFormula I, I′ or II, according to anyone of the previous embodiments,more specifically of anyone of embodiments 1 to 6, wherein X6 and X12are independently selected from K, Orn, Dab, E and D and wherein theside chain of X6 and X12 form together an amide bond; or an amide, anester or a salt of the peptide or polypeptide. In a further aspect ofthis embodiment, X6 is K, Orn or Dab and X12 is E or D and the sidechain of X6 and X12 form an amide bond. In yet another aspect of thisembodiment, X6 is K and X12 is E or D.

In embodiment 8, the invention pertains to a peptide or polypeptide ofFormula I, I′ or II, according to anyone of the previous embodiments,more specifically of anyone of embodiments 1 to 6, wherein X6 and X12are independently C, c, D-hC or hC wherein the side chain of X6 and X12form together a disulfide bond; or an amide, an ester or a salt of thepeptide or polypeptide. In a further aspect of this embodiment, X6 andX12 are C.

In embodiment 8A, the invention pertains to a peptide or polypeptide ofFormula I, I′ or II, according to anyone of the previous embodiments,more specifically of anyone of embodiments 1 to 6, wherein X6 and X12are independently C, c, D-hC or hC wherein the side chain of X6 and X12form together a monosulfide (—S—) bond; or an amide, an ester or a saltof the peptide or polypeptide. In a further aspect of this embodiment,X6 and X12 are C.

In embodiment 8B, the invention pertains to a peptide or polypeptide ofFormula I, I′ or II, according to anyone of the previous embodiments,more specifically of anyone of embodiments 1 to 6, wherein X6 and X12are independently C, c, D-hC or hC wherein the side chain of X6 and X12form together a linkage moiety of Formula —S—CH₂—C(═Z)—CH₂—S—; wherein═Z is ═O, =N—O—CH₂C(O)-L¹-R¹ or ═N—NH—CH₂C(O)-L¹-R¹ wherein R¹ is afatty acid derivative and L¹ is a linker containing one or more aminoacids and/or one or more —NH—C₂₋₆-alkylene-NH— units; or an amide, anester or a salt of the peptide or polypeptide. In a further aspect ofthis embodiment, X6 and X12 are C.

L1 is a suitable organic linker that connects a fatty acid derivative tothe polypeptide via an ester or an amide bond. Typically the linkercontains one or more amino acid moieties such as for example (O2Oc) unitor Glutamic acid, or contains —NH—C₂₋₆alkylene-NH— diamino units orcombination thereof. R¹ is a property-modifying group such as fatty acidderivative which can be added to increase the half-life of thepolypeptide. In one aspect of this embodiment, R1 is a fatty acid ofFormula C_(q)H_(q+1)C(O)₂H wherein q is 3 to 20. Optionally the terminalCH₃ unit may be oxidized to its corresponding carboxylic acid.

In one particular embodiment, -L¹-R¹ is of Formula:

q is an integer of 3 to 20. Preferably, q is an integer of 12 to 18.

A peptide or polypeptide of Formula I, I′ or II wherein X6 and X12 areindependently C, c, D-hC or hC wherein the side chain of X6 and X12 formtogether a linkage moiety of Formula —S—CH₂—C(═Z)—CH₂—S—; wherein ═Z is═O, =N—O—CH₂C(O)-L¹-R¹ or ═N—NH—CH₂C(O)-L¹-R¹ can be synthesizedaccording to Scheme 1:

Reduction of the polypeptide disulfide bond in (1A) is followed by anucleophilic reaction di-electrophilic compound (1B) forming polypeptide(1C). The ketone functionality of the polypeptide (1C) condensed readilywith an oxiamine or hydrazine to generate polypeptide 1D.

In embodiment 9, certain polypeptides of the invention include peptidesor polypeptides according to anyone of embodiment 1 to 6 and 8, havingFormula III:

or an amide, an ester or a salt of the polypeptide. In embodiment 9A,the invention pertains to peptides and polypeptides of Formula IIIwherein the 2 cysteine at position 6 and 12 form a disulfide bond(—S—S—), a monosulfide bond (—S—) or a linkage of Formula—S—CH₂—C(═Z)—CH₂—S— wherein Z is as defined supra. In a further aspectof embodiment 9 or 9A, the invention includes peptides or polypeptidesof Formula III wherein the 2 cysteines in position 6 and 12 form adisulfide bond (—S—S—).

In embodiment 10, certain peptides and polypeptides of the inventioninclude peptides and polypeptides according to anyone of embodiment 1-9having Formula IV:

or an amide, an ester or a salt of the polypeptide.

In embodiment 11, certain polypeptides of the invention includepolypeptides according to anyone of embodiment 1-6, and 8-10, havingFormula V:

or an amide, an ester or a salt of the polypeptide. In embodiment 11A,the invention pertains to peptides and polypeptides of Formula V whereinthe 2 cysteine at position 6 and 12 form a disulfide bond (—S—S—), amonosulfide bond (—S—) or a linkage of Formula —S—CH₂—C(═Z)—CH₂—S—wherein Z is as defined supra. In a further aspect of embodiment 11 or11A, the invention includes peptides or polypeptides of Formula Vwherein the 2 cysteines in position 6 and 12 form a disulfide bond(—S—S—).

In embodiment 12, the invention pertains to bicyclic peptides orpolypeptides of Formula I or I′ (according to embodiment 1, 2 or 3)wherein X3 is cysteine and wherein the side chain of cysteine forms adisulfide bond with the side chain of the cysteine at the X7 position.This embodiment is represented by peptides and polypeptides of FormulaVI:

or an amide, an ester or a salt of the polypeptide.

In embodiment 13, the invention pertains to bicyclic peptides orpolypeptides of Formula I or I′ (according to embodiment 1, 2 or 3)wherein X4 is cysteine and wherein the side chain of cysteine forms adisulfide bond with the side chain of the cysteine at the X7 position.This embodiment is represented by peptides and polypeptides of FormulaVII:

or an amide, an ester or a salt of the polypeptide.

In embodiment 14, the invention pertains to a peptide or polypeptide ofanyone of Formulae I to V, according to anyone of embodiment 1 to 11;wherein the N-terminus and the C-terminus optionally form a ringtogether with 1, 2, 3 or 4 glycine amino acids; or an amide, an ester ora salt of the polypeptide; or a polypeptide substantially equivalentthereto. This embodiment is represented by peptide or polypeptide havingFormula VIII:

wherein L is (G)r, G is glycine and r is 1, 2, 3 or 4; or a salt of thepolypeptide.

In embodiment 15, a further aspect of embodiment 14, the inventionpertains to peptide or polypeptide of Formula VIII wherein X1 is Q, X13is F and r is 2 or an ester, an amide or a salt thereof.

In embodiment 16, the invention pertains to a peptide or polypeptideaccording to Formula I or I′, according to embodiment 1, 2 or 3, whereinX6 is K, X13 is absent and X12 is F or f wherein the C-terminus of X12forms an amide bond with the amino side chain of X6. This embodiment isrepresented by a peptide or polypeptide of Formula IX:

or an ester, an amide or a salt of the polypeptide.

Any of the or below above-listed amino acid residues of Formula I′, orits related formulae and all embodiments described herein, e.g.,Formulae I, II to IX, may be substituted in a conservative fashion,provided the peptide or polypeptide of the invention still retainsfunctional activity and structural properties (e.g., half-lifeextension, protection from degradation, conformational constraint).Principle and examples of permissible conservative amino acidsubstitutions are further explained herein.

The following embodiments can be used independently, collectively or inany combination or sub-combination:

In embodiment 17, the invention pertains to peptides and polypeptidesaccording to any one of Formulae I′, I to VII and IX, or any of anyother classes and subclasses described supra, (i.e. according to anyoneof the embodiments 1 to 13 and 16) or an amide, an ester or a saltthereof, wherein X1 is pE.

In embodiment 18, the invention pertains to peptides and polypeptidesaccording to any one of Formulae I to VII and IX, or any of any otherclasses and subclasses described supra, (i.e. according to anyone of theembodiments 1 to 13 and 16) or an amide, an ester or a salt thereof,wherein X1 is absent; or an amide, an ester or a salt of thepolypeptide. In one aspect of this embodiment, the N-terminus of thepeptide is an amide. In embodiment 19, a further aspect of embodiment18, the invention pertains to peptides and polypeptide according to anyone of Formulae I to VII and IX, or any of any other classes andsubclasses described supra, or an amide, an ester or a salt thereof,wherein the X1 is absent and the N-terminus is an amide of Formula —NHRand R is CH₃C(O)—, CH₃—(O—CH₂CH₂)_(m)—C(O)—, Palmitoyl(O2Oc)_(p),Myristoyl(O2Oc)_(p), Lauroyl(O2Oc)_(p) or Ph-CH₂CH₂NHC(O)—, Acetyl,benzoyl, phenacyl, succinyl, octanoyl, 4-phenylbutanoyl,4-Cl-Ph-(CH2)₃C(O)—, or Ph-CH₂CH₂NHC(O)—; and wherein

p is an integer 1 to 4;m is an integer 1 to 12;Lauroyl(O2Oc)_(p) is C₁₁H₂₃C(O)[NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)]_(p)—;Myristoyl(O2Oc)_(p) is C₁₃H₂₇C(O)[NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)]_(p)—;Palmitoyl(O2Oc)_(p) is C₁₅H₃₁C(O)[NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)]_(p)—.In one particular aspect of this embodiment R is Acetyl, benzoyl,phenacyl, succinyl, octanoyl, 4-phenylbutanoyl, 4-Cl-Ph-(CH2)3C(O)—, orPh-CH₂CH₂NHC(O)—.

In embodiment 20, the invention pertains to peptide or polypeptideaccording to anyone of Formulae I to VII and IX, or any other classesand subclasses described supra, (i.e. according to anyone of embodiments1-13 and 16), or an amide, an ester or a salt thereof, whereinN-terminus is an amide of Formula NHR1 wherein R1 is CH₃C(O)—,CH₃—(O—CH₂CH₂)_(m)—C(O)—, Palmitoyl(O2Oc), Myristoyl(O2Oc),Lauroyl(O2Oc) or Ph-CH₂CH₂NHC(O)—; and wherein m, Lauroyl(O2Oc),Myristoyl(O2Oc) and Palmitoyl(O2Oc) are defined supra.

In embodiment 21, the invention pertains to peptides and polypeptidesaccording to any one of Formulae I to VII, or any of any other classesand subclasses described supra, (i.e. according to anyone of embodiments1 to 13), or an amide, an ester or a salt thereof, wherein X13 is F; oran amide, an ester or a salt of the polypeptide.

In embodiment 22, the invention pertains to peptides and polypeptidesaccording to any one of Formulae I to VII, or any of any other classesand subclasses described supra, (i.e. according to anyone of embodiments1 to 13), or an amide, an ester or a salt thereof, wherein X13 isabsent; or an amide, an ester or a salt of the polypeptide. Inembodiment 23, one aspect of embodiment 22, The C-terminus is an amide.In embodiment 24, a further aspect of embodiment 23, the inventionpertains to peptides and polypeptide according to any one of Formulae Ito VII, or any of any other classes and subclasses described supra, oran amide, an ester or a salt thereof, wherein the C-terminus is an amideof Formula —C(O)R2 and R2 is —NH₂, —NH-Me, —NH—NHBn, or —NH—(CH₂)₂-Ph.In a preferred aspect of embodiment 23, the invention pertains topeptides and polypeptide according to any one of Formulae I to VII, orany of any other classes and subclasses described supra, or an amide, anester or a salt thereof, wherein the C-terminus is an amide of Formula—C(O)R2 and R2 is —NH—(CH₂)₂-Ph.

In embodiment 25, the invention pertains to peptides and polypeptidesaccording to anyone of Formulae I to IX, or any of any other classes andsubclasses described supra, (i.e. according to anyone of embodiments 1to 25), or an amide, an ester or a salt thereof, wherein X5 is L.

In embodiment 26, the invention pertains to peptides and polypeptidesaccording to anyone of Formulae I to V, VIII and IX, or any of any otherclasses and subclasses described supra (i.e. according to anyone ofembodiments 1 to 11 and 14-25), or an amide, an ester or a salt thereof,wherein X7 is H.

In embodiment 27, the invention pertains to peptides and polypeptidesaccording to anyone of Formulae I o III and VI to IX, or any of anyother classes and subclasses described supra, (i.e. according to anyoneof embodiments 1 to 9 and 12 to 26), or an amide, an ester or a saltthereof, wherein X8 is K or F. In a further aspect of this embodiment,X8 is K.

In embodiment 28, the invention pertains to peptides and polypeptidesaccording to any one of Formulae I o III and VI to IX, or any of anyother classes and subclasses described supra, (i.e. according to anyoneof embodiments 1 to 9 and 12 to 27) or an amide, an ester or a saltthereof, wherein X9 is G.

In embodiment 29, the invention pertains to peptides and polypeptidesaccording to any one of Formulae I to IX, or any of any other classesand subclasses described supra, (i.e. according to anyone of embodiments1 to 28), or an amide, an ester or a salt thereof, wherein X11 is Nle.

In embodiment 30, the invention therefore provides a peptide or apolypeptide formula (I):

wherein:X1 is the N-terminus of the polypeptide and is either absent or isselected from pE, R, Isn and Q;

X2 is R, r, F or E;

X3 is P, p, K, D or 4-PhF; or cysteine wherein the side chain ofcysteine forms a disulfide bond with the side chain of the cysteine atthe X7 position;X4 is R, F, E or cysteine wherein the side chain of cysteine forms adisulfide bond with the side chain of the cysteine at the X7 position;

X5 is L, K, D or 4-PhF;

X6 and X12 are independently a natural or unnatural amino acid selectedfrom C, c, hC, D-hC, K, D, Orn, Dab or E wherein the side chain of X6and X12 are linked together via a covalent bond forming either adisulfide or an amide bond;or alternatively X6 is K, X13 is absent and X12 is F or f wherein theC-terminus of X12 form an amide bond with the amino side chain of X6;X7 is H, Aib, K, E, F or cysteine wherein the side chain of the cysteineforms a disulfide bond with the side chain of the cysteine at positionX3 or with the side chain of the cysteine at position X4;X8 is K, E, F or 4-amino-Isn;

X9 is G, D, L, R or Aib;

X10 is P or pipecolic acid,

X11 is Nle or 3-PyA; and

X13 is the C-terminus and is absent or is selected from F, f, K, H andE;wherein the N-terminus and the C-terminus optionally form a ringtogether with 1, 2, 3 or 4 glycine amino acids; andwherein the amino group in the side chain of K, Orn, Dab, Dap, hK or4-amino-Isn is optionally linked to a lipophilic group via an amidebond;or an amide, an ester or a salt of the polypeptide; or a polypeptidesubstantially equivalent thereto.

In embodiment 31, the invention pertains to a peptide or polypeptide ofembodiment 1, 2 or 3, wherein three of the amino acids X1 to X13 aredifferent from the corresponding amino acids present in Pyr-1-apelin-13.In embodiment 32, the invention pertains to a peptide or polypeptide ofembodiment 1, 2 or 3 wherein four of the amino acids X1 to X13 aredifferent from the corresponding amino acids present in Pyr-1-apelin-13.

In another embodiment, X1, X2, X3, X4, X5, X6, X7, X8. X9, X10, X11, X12and X13 amino acids are those defined by X1, X2, X3, X4, X5, X6, X7, X8.X9, X10, X11, X12 and X13 amino acids in the Examples section below.

In another embodiment, individual polypeptides according to theinvention are those listed in the Examples section below or apharmaceutically acceptable salt thereof.

Unless specified otherwise, the term “polypeptide of the presentinvention” refers to a polypeptide of Formula (I′) and subformulaethereof (Formulae I, II to IX); or an amide, an ester or a salt thereof.

Unless specified otherwise, the terms “polypeptides of the presentinvention,” “peptides of the present invention,” “apelin peptideagonists,” and the like refer to peptides and polypeptides of Formula I′and subformulae thereof (Formulae I, II, III, IV, V, VI, VII, VIII orIX); or an amide, an ester or a salt thereof. The peptides andpolypeptides of the invention demonstrate substantially equivalent orimproved activity and/or plasma stability over known apelin peptides andpolypeptides described herein, including but not limited to wild typeapelin, apelin-13 and pyr-1-apelin-13.

The peptides and polypeptides of the invention also encompass peptidesand polypeptides that are at least about 95% identical to the peptidesand polypeptides according to any one of Formulae I′, I to IX, or anamide, an ester or a salt thereof, as well as to any peptides orpolypeptides specifically listed herein, including but not limited tothe experimental examples.

As used herein, the phrase “homologous amino acid sequence,” orvariations thereof, refers to sequences characterized by a homology, atthe amino acid level, of at least a specified percentage and is usedinterchangeably with “sequence identity.” Homologous amino acidsequences include those amino acid sequences which contain conservativeamino acid substitutions and which polypeptides have the same bindingand/or activity. In some embodiments, an amino acid sequence ishomologous if it has at least 60% or greater, up to 99%, identity with acomparator sequence. In some embodiments, an amino acid sequence ishomologous if it shares one or more, up to 60, amino acid substitutions,additions, or deletions with a comparator sequence. In some embodiments,the homologous amino acid sequences have no more than 5 or no more than3 conservative amino acid substitutions.

Homology may also be at the polypeptide level. The degree or percentageidentity of peptides or polypeptides of the invention, or portionsthereof, and different amino acid sequences is calculated as the numberof exact matches in an alignment of the two sequences divided by thelength of the “invention sequence” or the “foreign sequence”, whicheveris shortest. The result is expressed as percent identity.

A polypeptide comprising an amino acid sequence having a homology ofabout 80-99.9%, preferably 90-99.9% to the amino acid sequence describedin the specific examples, and possessing a plasma stability superior toapelin-13 or pyr-1-apelin-13, fall under the category of the polypeptideof the invention. In one embodiment, the plasma stability improvement isat least 2 fold. In one embodiment, the polypeptide of the invention hasa plasma stability of at least 30 minutes. In another embodiment, thepolypeptide of the invention has a plasma stability of at least 60minutes, preferably at least 100 min and more preferably at least 150minutes.

The term “substantially equivalent” means the nature of thereceptor-binding activity, signal transduction activity and the like isequivalent. Thus, it is allowable that even differences among gradessuch as the strength of receptor binding activity and the molecularweight of the polypeptide are present.

A polypeptide as described herein, or a substantial equivalent thereto,by substitution, deletion, addition or insertion of one or more of aminoacids may be mentioned as polypeptides containing an amino acid sequencesubstantial equivalent(s) in the above sense. A polypeptide as describedherein, or a substantial equivalent thereto, by substitution of 1 to 5,preferably 1 to 3 and more preferably 1 or 2 amino acids with natural orun-natural amino acids may be mentioned as polypeptides containing anamino acid sequence substantial equivalent(s) in the above sense.Further modifications and alterations may include the replacement of anL-amino-acid with a D-amino acid, or other variation including, but notlimited to, phosphorylation, carboxylation, alkylation and the like aslong as the apelin agonistic activity of the peptide of polypeptide ofFormulae I, II, III, IV, V, VI, VII, VIII or IX is maintained and theplasma stability is improved over the pyroglutamated form of apelin-13.For example, D-amino acid are well tolerated with respect to activityand stability of the polypeptide at position 2 (X2), position 3 (X3),positions 5, 6, 7 and 8 (X5, X6, X7 and X8), position 10 (X10) andposition 13 (X13) of the cyclic peptides and polypeptides of Formulae I,II, III, IV, V, VI, VII, VIII or IX.

As used herein, the term “pharmaceutically acceptable salts” refers tosalts that retain the biological effectiveness and properties of thepolypeptides of this invention and, which typically are not biologicallyor otherwise undesirable. In many cases, the polypeptides of the presentinvention are capable of forming acid and/or base salts by virtue of thepresence of amino and/or carboxyl groups or groups similar thereto.

Pharmaceutically acceptable acid addition salts can be formed withinorganic acids and organic acids, e.g., acetate, aspartate, benzoate,besylate, bromide/hydrobromide, bicarbonate/carbonate,bisulfate/sulfate, camphorsulfornate, chloride/hydrochloride,chlortheophyllonate, citrate, ethandisulfonate, fumarate, gluceptate,gluconate, glucuronate, hippurate, hydroiodide/iodide, isethionate,lactate, lactobionate, laurylsulfate, malate, maleate, malonate,mandelate, mesylate, methylsulphate, naphthoate, napsylate, nicotinate,nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate,phosphate/hydrogen phosphate/dihydrogen phosphate, polygalacturonate,propionate, stearate, succinate, sulfosalicylate, tartrate, tosylate andtrifluoroacetate salts.

Inorganic acids from which salts can be derived include, for example,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Organic acids from which salts can bederived include, for example, acetic acid, propionic acid, glycolicacid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid,sulfosalicylic acid, and the like. Pharmaceutically acceptable baseaddition salts can be formed with inorganic and organic bases.

Inorganic bases from which salts can be derived include, for example,ammonium salts and metals from columns I to XII of the periodic table.In certain embodiments, the salts are derived from sodium, potassium,ammonium, calcium, magnesium, iron, silver, zinc, and copper;particularly suitable salts include ammonium, potassium, sodium, calciumand magnesium salts.

Organic bases from which salts can be derived include, for example,primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines, basic ionexchange resins, and the like. Certain organic amines includeisopropylamine, benzathine, cholinate, diethanolamine, diethylamine,lysine, meglumine, piperazine and tromethamine.

The pharmaceutically acceptable salts of the present invention can besynthesized from a parent compound, a basic or acidic moiety, byconventional chemical methods. Generally, such salts can be prepared byreacting free acid forms of these compounds with a stoichiometric amountof the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate,bicarbonate or the like), or by reacting free base forms of thesecompounds with a stoichiometric amount of the appropriate acid. Suchreactions are typically carried out in water or in an organic solvent,or in a mixture of the two. Generally, use of non-aqueous media likeether, ethyl acetate, ethanol, isopropanol, or acetonitrile isdesirable, where practicable. Lists of additional suitable salts can befound, e.g., in “Remington's Pharmaceutical Sciences”, 20th ed., MackPublishing Company, Easton, Pa., (1985); and in “Handbook ofPharmaceutical Salts: Properties, Selection, and Use” by Stahl andWermuth (Wiley-VCH, Weinheim, Germany, 2002).

As used herein, the term “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, surfactants,antioxidants, preservatives (e.g., antibacterial agents, antifungalagents), isotonic agents, absorption delaying agents, salts,preservatives, drugs, drug stabilizers, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, and the like and combinations thereof, as would be known to thoseskilled in the art (see, for example, Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329). Exceptinsofar as any conventional carrier is incompatible with the activeingredient, its use in the therapeutic or pharmaceutical compositions iscontemplated.

The term “a therapeutically effective amount” of a polypeptide of thepresent invention refers to an amount of the polypeptide of the presentinvention that will elicit the biological or medical response of asubject, for example, amelioration of a symptom, alleviation of acondition, slow or delay disease progression, or prevention of adisease, etc. In one non-limiting embodiment, the term “atherapeutically effective amount” refers to the amount of thepolypeptide of the present invention that, when administered to asubject, is effective to (1) at least partially alleviate, inhibit,prevent and/or ameliorate a condition, a disorder or a disease or asymptom thereof (D ameliorated by the activation of the APJ receptor or(ii) associated with the activity of the APJ receptor, or (iii)characterized by abnormal activity of the APJ receptor; or (2) activatethe APJ receptor.

In another non-limiting embodiment, the term “a therapeuticallyeffective amount” refers to the amount of the polypeptide of the presentinvention that, when administered to a cell, or a tissue, or anon-cellular biological material, or a medium, is effective to at leastpartially activate the APJ receptor. As will be appreciated by those ofordinary skill in the art, the absolute amount of a particular agentthat is effective may vary depending on such factors as the desiredbiological endpoint, the agent to be delivered, the target tissue, etc.Those of ordinary skill in the art understand that “a therapeuticallyeffective amount” may be administered in a single dose or may beachieved by administration of multiple doses. For example, in the caseof an agent to treat heartfailure, an effective amount may be an amountsufficient to result in clinical improvement of the patient, e.g.,increased exercise tolerance/capacity, increased blood pressure,decrease fluid retention, and/or improved results on a quantitative testof cardiac functioning, e.g., ejection fraction, exercise capacity (timeto exhaustion), etc.

As used herein, the term “subject” refers to an animal. Typically theanimal is a mammal. A subject also refers to for example, primates(e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats,mice, fish, birds and the like. In certain embodiments, the subject is aprimate. In yet other embodiments, the subject is a human.

As used herein, the term “inhibit”, “inhibition” or “inhibiting” refersto the reduction or suppression of a given condition, symptom, ordisorder, or disease, or a significant decrease in the baseline activityof a biological activity or process.

As used herein, the term “treat”, “treating” or “treatment” of anydisease or disorder refers in one embodiment, to ameliorating thedisease or disorder (i.e., slowing or arresting or reducing thedevelopment of the disease or at least one of the clinical symptomsthereof). In another embodiment “treat”, “treating” or “treatment”refers to alleviating or ameliorating at least one physical parameterincluding those which may not be discernible by the patient. In yetanother embodiment, “treat”, “treating” or “treatment” refers tomodulating the disease or disorder, either physically, (e.g.,stabilization of a discernible symptom), physiologically, (e.g.,stabilization of a physical parameter), or both. In yet anotherembodiment, “treat”, “treating” or “treatment” refers to preventing ordelaying the onset or development or progression of the disease ordisorder.

As used herein, the terms “prevent”, “preventing” and “prevention” referto the prevention of the recurrence, onset, or development of one ormore symptoms of a disorder in a subject resulting from theadministration of a therapy (e.g., a therapeutic agent), or theadministration of a combination of therapies (e.g., a combination oftherapeutic agents).

As used herein, a subject is “in need of” a treatment if such subjectwould benefit biologically, medically or in quality of life from suchtreatment.

As used herein, the term “a,” “an,” “the” and similar terms used in thecontext of the present invention (especially in the context of theclaims) are to be construed to cover both the singular and plural unlessotherwise indicated herein or clearly contradicted by the context.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.“such as”) provided herein is intended merely to better illuminate theinvention and does not pose a limitation on the scope of the inventionotherwise claimed.

The peptides and polypeptides of the present invention can be producedby the per se known procedures for peptide synthesis. The methods forpeptide synthesis may be any of a solid-phase synthesis and aliquid-phase synthesis. Thus, the peptide and polypeptide of interestcan be produced by condensing a partial peptide or amino acid capable ofconstituting the protein with the residual part thereof and, when theproduct has a protective group, the protective group is detachedwhereupon a desired peptide can be manufactured. The known methods forcondensation and deprotection include the procedures described in thefollowing literature (1)-(5).

-   -   (1) M. Bodanszky and M. A. Ondetti, Peptide Synthesis,        Interscience Publishers, New York, 1966,    -   (2) Schroeder and Luebke, The Peptide, Academic Press, New York,        1965,    -   (3) Nobuo Izumiya et al. Fundamentals and Experiments in Peptide        Synthesis, Maruzen, 1975,    -   (4) Haruaki Yajima and Shumpei Sakakibara, Biochemical        Experiment Series 1, Protein Chemistry IV, 205, 1977, and    -   (5) Haruaki Yajima (ed.), Development of Drugs-Continued, 14,        Peptide Synthesis, Hirokawa Shoten.

After the reaction, the peptide can be purified and isolated by acombination of conventional purification techniques such as solventextraction, column chromatography, liquid chromatography, andrecrystallization. Where the peptide isolated as above is a freecompound, it can be converted to a suitable salt by the known method.Conversely where the isolated product is a salt, it can be converted tothe free peptide by the known method.

The amide of polypeptide can be obtained by using a resin for peptidesynthesis which is suited for amidation. The resin includes chloromethylresin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin,4-benzyloxybenzyl alcohol resin, 4-methylbenz-hydrylamine resin, PAMresin, 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamideresin, 4-(2′,4′-dimethoxyphenyl-hydroxymethyl)phenoxy resin,4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin, 2-chlorotritylchloride resin, and so on. Using such a resin, amino acids whose α-aminogroups and functional groups of side-chain have been suitably protectedare condensed on the resin according to the sequence of the objectivepeptide by various condensation techniques which are known per se. Atthe end of the series of reactions, the peptide or the protected peptideis removed from the resin and the protective groups are removed and ifnecessary, disulfide bonds are formed to obtain the objectivepolypeptide.

For the condensation of the above-mentioned protected amino acids, avariety of activating reagents for peptide synthesis can be used such asHATU, HCTU or e.g. a carbodiimide. The carbodiimide includes DCC,N,N′-diisopropylcarbodiimide, andN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide. For activation with sucha reagent, a racemization inhibitor additive, e.g. HOBt or Oxyma Purecan be used. The protected amino acid can be directly added to the resinalong with the activation reagents and racemization inhibitor or bepre-activated as symmetric acid anhydride, HOBt ester, or HOOBt esterthen added to the resin. The solvent for the activation of protectedamino acids or condensation with the resin can be properly selected fromamong those solvents which are known to be useful for peptidecondensation reactions. For example, N,N-dimethylformamide,N-methylpyrrolidone, chloroform, trifluoroethanol, dimethyl sulfoxide,DMF, pyridine, dioxane, methylene chloride, tetrahydrofuran,acetonitrile, ethyl acetate, or suitable mixtures of them can bementioned.

The reaction temperature can be selected from the range hitherto-knownto be useful for peptide bond formation and is usually selected from therange of about −20° C.-50° C. The activated amino acid derivative isgenerally used in a proportion of 1.5-4 fold excess. If the condensationis found to be insufficient by a test utilizing the ninhydrin reaction,the condensation reaction can be repeated to achieve a sufficientcondensation without removing the protective group. If repeatedcondensation still fails to provide a sufficient degree of condensation,the unreacted amino group can be acetylated with acetic anhydride oracetylimidazole.

The protecting group of amino group for the starting material amino acidincludes Z, Boc, tertiary-amyloxycarbonyl, isobornyloxycarbonyl,4-methoxybenzyloxycarbonyl, Cl—Z, Br—Z, adamantyloxycarbonyl,trifluoroacetyl, phthalyl, formyl, 2-nitrophenylsulfenyl,diphenylphosphinothioyl, or Fmoc. The carboxy-protecting group that canbe used includes but is not limited to the above-mentioned C₁₋₆ alkyl,C₃₋₈ cycloalkyl and C₆₋₁₀aryl-C₁₋₂alkyl as well as 2-adamantyl,4-nitrobenzyl, 4-methoxybenzyl, 4-chlorobenzyl, phenacyl,benzyloxycarbonylhydrazido, tertiary-butoxycarbonylhydrazido, andtritylhydrazido.

The hydroxy group of serine and threonine can be protected byesterification or etherification. The group suited for saidesterification includes carbon-derived groups such as lower alkanoylgroups, e.g. acetyl etc., aroyl groups, e.g. benzoyl etc.,benzyloxycarbonyl, and ethoxycarbonyl. The group suited for saidetherification includes benzyl, tetrahydropyranyl, and tertiary-butyl.The protective group for the phenolic hydroxyl group of tyrosineincludes Bzl, Cl₂-Bzl, 2-nitrobenzyl, Br—Z, and tertiary-butyl.

The protecting group of imidazole for histidine includes Tos,4-methoxy-2,3,6-tri ethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum,Boc, Trt, and Fmoc.

The activated carboxyl group of the starting amino acid includes thecorresponding acid anhydride, azide and active esters, e.g. esters withalcohols such as pentachlorophenol, 2,4,5-trichlorophenol,2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, HONB,N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt, etc. The activatedamino group of the starting amino acid includes the correspondingphosphoramide.

The method for elimination of protective groups includes catalyticreduction using hydrogen gas in the presence of a catalyst such aspalladium black or palladium-on-carbon, acid treatment with anhydroushydrogen fluoride, methanesulfonic acid, trifluoromethanesuifonic acid,trifluoroacetic acid, or a mixture of such acids, base treatment withdiisopropylethylamine, triethylamine, piperidine, piperazine, reductionwith sodium metal in liquid ammonia. The elimination reaction by theabove-mentioned acid treatment is generally carried out at a temperatureof −20° C.-40° C. and can be conducted advantageously with addition of acation acceptor such as anisole, phenol, thioanisole, m-cresol,p-cresol, dimethyl sulfide, 1,4-butanedithiol, 1,2-ethanedithiol. The2,4-dinitrophenyl group used for protecting the imidazole group ofhistidine can be eliminated by treatment with thiophenol, while theformyl group used for protecting the indole group of tryptophan can beeliminated by alkali treatment with dilute sodium hydroxide solution ordilute aqueous ammonia as well as the above-mentioned acid treatment inthe presence of 1,2-ethanedithiol, 1,4-butanedithiol.

The method for protecting functional groups which should not take partin the reaction of the starting material, the protective groups that canbe used, the method of removing the protective groups, and the method ofactivating the functional groups that are to take part in the reactioncan all be selected judicially from among the known groups and methods.

An another method for obtaining the amide form of the polypeptidecomprises amidating the -carboxyl group of the C-terminal amino acid atfirst, then extending the peptide chain to the N-side until the desiredchain length, and then selectively deprotecting the α-amino group of theC-terminal peptide and the α-carboxy group of the amino acid or peptidethat is to form the remainder of the objective polypeptide andcondensing the two fragments whose α-amino group and side-chainfunctional groups have been protected with suitable protective groupsmentioned above in a mixed solvent such as that mentioned hereinbefore.The parameters of this condensation reaction can be the same asdescribed hereinbefore. From the protected peptide obtained bycondensation, all the protective groups are removed by theabove-described method to thereby provide the desired crude peptide.This crude peptide can be purified by known purification procedures andthe main fraction be lyophilized to provide the objective amidatedpolypeptide. To obtain an ester of the polypeptide, the α-carboxyl groupof the C-terminal amino acid is condensed with a desired alcohol to givean amino acid ester and then, the procedure described above forproduction of the amide is followed.

The polypeptides of the instant invention, or an amide, an ester of asalt thereof, may be administered in any of a variety of ways, includingsubcutaneously, intramuscularly, intravenously, intraperitoneally,inhalationally, etc. Particularly preferred embodiments of the inventionemploy continuous intravenous administration of the polypeptides of theinstant invention, or an amide, ester, or salt thereof. The polypeptideson the instant invention may be administered as a bolus or as acontinuous infusion over a period of time. An implantable pump may beused. In certain embodiments of the invention, intermittent orcontinuous polypeptides administration is continued for one to severaldays (e.g., 2-3 or more days), or for longer periods of time, e.g.,weeks, months, or years. In some embodiments, intermittent or continuouspolypeptide administration is provided for at least about 3 days. Inother embodiments, intermittent or continuous polypeptide administrationis provided for at least about one week. In other embodiments,intermittent or continuous polypeptide administration is provided for atleast about two weeks. It may be desirable to maintain an average plasmapolypeptide concentration above a particular threshold value eitherduring administration or between administration of multiple doses. Adesirable concentration may be determined, for example, based on thesubject's physiological condition, disease severity, etc. Such desirablevalue(s) can be identified by performing standard clinical trials.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a polypeptide of the present invention or andamide, an ester or a salt thereof and one or more pharmaceuticallyacceptable carriers. The pharmaceutical composition can be formulatedfor particular routes of administration such as oral administration,parenteral administration, and rectal administration, etc. In addition,the pharmaceutical compositions of the present invention can be made upin a solid form (including without limitation capsules, tablets, pills,granules, lyophilizates, powders or suppositories), or in a liquid form(including without limitation solutions, suspensions or emulsions). Thepharmaceutical compositions can be subjected to conventionalpharmaceutical operations such as aseptic manufacturing, sterilizationand/or can contain conventional inert diluents, cake forming agents,tonicity agents, lubricating agents, or buffering agents, as well asadjuvants, such as preservatives, stabilizers, wetting agents,emulsifers and buffers, etc.

Pharmaceutical compositions suitable for injectable use typicallyinclude sterile aqueous solutions (where water soluble) or dispersionsand sterile powders for the extemporaneous preparation of sterileinjectable solutions or dispersion.

For intravenous administration, suitable carriers include physiologicalsaline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). In all cases, the composition should besterile and should be fluid to the extent that easy syringabilityexists. Preferred pharmaceutical formulations are stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Ingeneral, the relevant carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like), andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, amino acids, sorbitol, sodiumchloride in the composition. Prolonged absorption of the injectablecompositions can be brought about by including in the composition anagent which delays absorption, for example, aluminum monostearate andgelatin.

Certain injectable compositions are aqueous isotonic solutions orsuspensions, and suppositories are advantageously prepared from fattyemulsions or suspensions. Said compositions may be sterilized and/orcontain adjuvants, such as preserving, stabilizing, wetting oremulsifying agents, solution promoters, salts for regulating the osmoticpressure and/or buffers. In addition, they may also contain othertherapeutically valuable substances. Said compositions are preparedaccording to conventional mixing, granulating or coating methods,respectively, and contain about 0.1-75%, or contain about 1-50%, of theactive ingredient.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltration sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. Formulations fororal delivery may advantageously incorporate agents to improve stabilitywithin the gastrointestinal tract and/or to enhance absorption.

For administration by inhalation, the inventive therapeutic agents arepreferably delivered in the form of an aerosol spray from pressuredcontainer or dispenser which contains a suitable propellant, e.g., a gassuch as carbon dioxide, or a nebulizer. It is noted that the lungsprovide a large surface area for systemic delivery of therapeuticagents.

The agents may be encapsulated, e.g., in polymeric microparticles suchas those described in U.S. publication 20040096403, or in associationwith any of a wide variety of other drug delivery vehicles that areknown in the art. In other embodiments of the invention the agents aredelivered in association with a charged lipid as described, for example,in U.S. publication 20040062718. It is noted that the latter system hasbeen used for administration of a therapeutic polypeptide, insulin,demonstrating the utility of this system for administration of peptideagents.

Systemic administration can also be by transmucosal or transdermalmeans.

Suitable compositions for transdermal application include an effectiveamount of a polypeptide of the invention with a suitable carrier.Carriers suitable for transdermal delivery include absorbablepharmacologically acceptable solvents to assist passage through the skinof the host. For example, transdermal devices are in the form of abandage comprising a backing member, a reservoir containing the compoundoptionally with carriers, optionally a rate controlling barrier todeliver the compound of the skin of the host at a controlled andpredetermined rate over a prolonged period of time, and means to securethe device to the skin.

Suitable compositions for topical application, e.g., to the skin andeyes, include aqueous solutions, suspensions, ointments, creams, gels orsprayable formulations, e.g, for delivery by aerosol or the like. Suchtopical delivery systems will in particular be appropriate for dermalapplication. They are thus particularly suited for use in topical,including cosmetic, formulations well-known in the art. Such may containsolubilizers, stabilizers, tonicity enhancing agents, buffers andpreservatives.

As used herein a topical application may also pertain to an inhalationor to an intranasal application. They may be conveniently delivered inthe form of a dry powder (either alone, as a mixture, for example a dryblend with lactose, or a mixed component particle, for example withphospholipids) from a dry powder inhaler or an aerosol spraypresentation from a pressurised container, pump, spray, atomizer ornebuliser, with or without the use of a suitable propellant.

The invention further provides pharmaceutical compositions and dosageforms that comprise one or more agents that reduce the rate by which thecompound of the present invention as an active ingredient willdecompose. Such agents, which are referred to herein as “stabilizers,”include, but are not limited to, antioxidants such as ascorbic acid, pHbuffers, or salt buffers, etc.

Method of the Invention:

Apelin family of peptides is the only known natural family of ligandsfor the G protein coupled APJ receptor. Apelin gene encodes a 77aminoacid polypeptide, which gets processed into biologically activeforms of apelin peptides, such as apelin-36, apelin-17, apelin-16,apelin-13, apelin-12 and pyroglutamate modified form of apelin-13(Pyr′-apelin-13). Any one of these apelin peptides, upon binding to APJreceptor, transduces the signal via Gi and Gq proteins. Incardiomyocytes, Gi or Gq coupling leads to changes in intracellular pH,PLC activation, and IP3 production that enhance myofilament calciumsensitivity and ultimately result in increased cardiac contractility. Gicoupling inhibits activated Gs, adenylyl cyclase and cAMP production andincreases pAkt levels leading to cardioprotection. In vascularendothelial cells, APJ activation via Gi, pAKT leads to increased nitricoxide (NO) production, which increases smooth muscle relaxationresulting in overall vasodilation.

Patients with chronic stable heart failure have occasional acuteepisodes of decompensation, where cardiac contractility declines furtherand symptoms worsen. These exacerbations are referred to as acutedecompensated heart failure (ADHF). Current therapies for ADHF includediuretics, vasodilators, and inotropes, which directly increase cardiaccontractility. Current intravenous inotropes (dobutamine, dopamine,milrinone, levosimendan) are well known for their adverse events such asarrhythmia and increased long-term mortality. The synthetic apelinpolypeptide analogs of the instant invention provide a therapy for ADHFthat increases cardiac contractility without arrhythmogenic or mortalityliabilities and address the enormous unmet medical need in chronic heartfailure.

Indeed, acute apelin treatment (5 min) in humans results in coronaryvasodilatation and improved cardiac output. However, native apelinsexhibit a very short t_(1/2) (seconds) and duration of action (fewminutes) in vivo. The potent synthetic apelin peptide agonists of theinstant invention have longer half lives compared to the native apelin.

Activation of APJ receptor in cardiomyocytes a) improve cardiaccontractility via Gi/Gq, PLC and Ca2+, and b) provide cardioprotectionvia Gi, pAkt activation, but without increasing cAMP (as seen with otherinotropes). In addition, APJ agonism in endothelial cells leads toarterial vasodilation, which further benefits heart failure by unloadingthe work of left ventricle. Taken together the synthetic apelinpolypeptide analogs can improve overall cardiac function, reducearrhythmogenesis and provide survival benefit.

More recently, there have been a number of preclinical researchpublications focusing on the potential involvement of Apelin in diabetesand insulin resistance. Apelin has been shown to 1) lower blood glucoselevels by improving glucose uptake in muscle, adipose and heart, 2)protect pancreatic beta cells from ER stress and subsequent apoptosis,3) lower the insulin secretion in beta cells, and 4) regulatecatecholamine induced lypolysis in adipose tissue. Activation of pAKTpathway has been implicated in these processes.

The polypeptides according to anyone of formulae I to IX, or apharmaceutically acceptable salt thereof, in free form or inpharmaceutically acceptable salt form, exhibit valuable pharmacologicalproperties, e.g. APJ receptor agonsim properties, e.g. as indicated inin vitro and in vivo tests as provided in the next sections and aretherefore indicated for therapy.

Polypeptides of the invention or a pharmaceutically acceptable saltthereof, may be useful in the treatment of an indication selected fromacute decompensated heart failure (ADHF), chronic heart failure,pulmonary hypertension, atrial fibrillation, Brugada syndrome,ventricular tachycardia, atherosclerosis, hypertension, restenosis,ischemic cardiovascular diseases, cardiomyopathy, cardiac fibrosis,arrhythmia, water retention, diabetes (including gestational diabetes),obesity, peripheral arterial disease, cerebrovascular accidents,transient ischemic attacks, traumatic brain injuries, amyotrophiclateral sclerosis, burn injuries (including sunburn) and preeclampsia.

Thus, as a further embodiment, the present invention provides the use ofa polypeptide of anyone of formulae I to IX, or an amide, an ester or asalt thereof for the treatment of a disease which is associated with theAPJ receptor activity. In a further embodiment, the therapy is selectedfrom a disease which is responsive to the agonism of the APJ receptor.In another embodiment, the disease is selected from the afore-mentionedlist, suitably acute decompensated heart failure. In yet another subsetof this embodiment, the present invention provides the use of apolypeptide of anyone of formulae I to IX, or an amide, ester or a saltthereof, in the manufacture of a medicament, for the treatment of adisease which is associated with the APJ receptor activity.

Thus, as a further embodiment, the present invention provides the use ofa polypeptide of anyone of formulae I to IX, or an amide, an ester or asalt thereof, in therapy. In a further embodiment, the therapy isselected from a disease which may be treated by activation (agonism) ofthe APJ receptor.

In another embodiment, the invention provides a method of treating adisease which is responsive to the agonism of the APJ receptor,comprising administration of a therapeutically acceptable amount of apolypeptide of anyone of formulae I to IX, or an amide, an ester of asalt thereof. In a further embodiment, the disease is selected from theafore-mentioned list, suitably acute decompensated heart failure.

In yet another subset of this embodiment, the invention provides amethod of treating a disease which is associated with the activity ofthe APJ receptor comprising administration of a therapeuticallyacceptable amount of a polypeptide of anyone of formulae I to IX, or anamide, an ester or a salt thereof.

The effective amount of a pharmaceutical composition or combination ofthe invention to be employed therapeutically will depend, for example,upon the therapeutic context and objectives. One skilled in the art willappreciate that the appropriate dosage levels for treatment will thusvary depending, in part, upon the molecule delivered, the indication forwhich the fusion protein variant is being used, the route ofadministration, and the size (body weight, body surface, or organ size)and condition (the age and general health) of the patient. Accordingly,the clinician can titer the dosage and modify the route ofadministration to obtain the optimal therapeutic effect. A typicaldosage can range from about 0.1 μg/kg to up to about 100 mg/kg or more,depending on the factors mentioned above. In other embodiments, thedosage can range from 0.1 μg/kg up to about 100 mg/kg; or 1 μg/kg up toabout 100 mg/kg.

The frequency of dosing will depend upon the pharmacokinetic parametersof the dual function protein in the formulation being used. Typically, aclinician will administer the composition until a dosage is reached thatachieves the desired effect. The composition can therefore beadministered as a single dose, as two or more doses (which may or maynot contain the same amount of the desired molecule) over time, or as acontinuous infusion via an implantation device or catheter. Furtherrefinement of the appropriate dosage is routinely made by those ofordinary skill in the art and is within the ambit of tasks routinelyperformed by them. Appropriate dosages can be ascertained through use ofappropriate dose-response data.

The activity of a polypeptide according to the present invention can beassessed by the following in vitro methods described below.

hAPJ Calcium Flux Assay:

Chem-5 APJ stable cells (Millipore # HTS068C) were plated in 384-wellformat with 10,000 cells/well in 25 ul growth media, then grown 24 hoursin a 37° C. tissue culture incubator. One hour before the assay, 25ul/well FLIPR Calcium 4 dye (Molecular Devices R8142) with 2.5 mMprobenecid was added, and cells were incubated one hour in a 37° C.tissue culture incubator. Peptides were solubilized in HBSS, HEPES &0.1% BSA buffer, and serially-diluted 10-fold, from 50 uM to 5 pM, intriplicate. FLIPR Tetra was used to add peptide to the cells with dye(1:5, for final peptide concentrations ranging from 10 uM to 1 pM).FLIPR dye inside the cells emitted fluorescence after binding tocalcium, while fluorescence from outside the cells was masked.Fluorescence was measured using 470-495 excitation and 515-575 emissionwavelengths on the FLIPR Tetra. Readings were done for 3 minutes total,beginning 10 seconds before the peptide addition. Maximum-minimum valueswere calculated and plotted for each peptide concentration, and GraphPadprism software was used to calculate EC₅₀ values at the curve inflectionpoints, for calcium flux stimulation by peptides.

Plasma Stability Assay: Materials:

Working solution: 1 mg/mL test article is prepared in Milli-Q waterExtraction solution: Methanol:Acetonitrile:Water (1:1:1) with 0.1%Formic Acid and 400 ng/mL Glyburide.Plasma: Male Sprague-Dawley rat plasma (with sodium heparin), purchasedfrom Bioreclamation LLC (Liverpool, N.Y.).Whole blood: Male Sprague Dawley whole blood (with sodium heparin),purchased from Bioreclamation LLC (Liverpool, N.Y.)Lung homogenate: Male rat Sprague Dawley lung was purchased fromBioreclamation LLC (Liverpool, N.Y.). The lung was homogenized usingpolytron homogenizer after addition of 5× volume of 1×PBS. Thehomogenate was centrifuged at 9000 rpm for 10 min at 4° C. Thesupernatant was centrifuged again at 3000 rpm for 30 min to make a clearsupernatant. Protein concentration was determined using a commercial kit(Pierce, Thermo Scientific).

Sample Preparation Procedure:

Test article was prepared in one of the following biological matrices:heparinized rat plasma, heparinized rat whole blood or lung homogenate.The plasma and whole blood sample was prepared at 5000 ng/mL by adding 5uL of 1 mg/mL Working solution to 995 uL of rat plasma or whole blood.Lung homogenate samples were prepared by diluting lung homogenate to 1mg/ml protein concentration with phosphate buffered saline (PBS),followed by addition of 5 uL Working solution to 995 uL diluted lunghomogenate. The samples were incubated at 37° C. with gentle shaking(65˜75 rpm) in a water bath incubator. At times 0 min, 5 min, 15 min, 30min, 60 min, 120 and 240 min, 25 uL aliquots of incubation samples weretransferred to 96-well plate and immediately protein precipitated using150 uL of Extraction solution. After completion of incubationexperiment, the sample plate was centrifuged at 4000 rpm at 4° C. for 10minutes. Afterwards, a pipetting device (Tecan Temo) was used totransfer the supernatants to another plate and add 50 uL of water to allsamples. The plate was vortexed prior to LC-MS analysis.

LC-MS Analysis of stability samplesHPLC: Agilent 1290 HPLC with autosamplerColumn: MAC-MOD ACE C18, 3 μm, 30 mm×2.1 mm i.d.Mobile phase A: 0.1% Formic acid in acetonitrileMobile phase B: 0.1% Formic acid in water

Gradient Program:

Mobile Mobile Time (min) Flow (mL) Phase A(%) Phase B(%) 0 0.4 95 5 0.50.4 95 5 1.5 0.4 5 95 4.1 0.4 5 95 4.2 0.4 95 5 5 0.4 95 5Mass spectrometer: Agilent Q-TOF 6530Data acquisition mode: Full scan with mass range of 100-1000 m/zData acquisition and analysis software: MassHunter

Data Analysis:

Stability assay: stability half-life, (t ½), values were determined byconverting peak areas at each time point to percent remaining relativeto initial (t=0) peak area.

Percent remaining=100×(sample peak area)÷(t=0 peak area)

The natural log of percent remaining values were calculated and plottedagainst sample time (Microsoft Excel). The slope of this line, k, wasdetermined by linear regression (Microsoft Excel).Stability half-life was then calculated by the formula, t ½=0.693÷k

Surrogate Activity-Based Plasma Stability Assay:

The calcium flux protocol described above was followed, with thefollowing changes. The peptides were also incubated with 5% rat plasma(Bioreclamation # RATPLNAHP-M, Na Heparin-treated). Readings were takenat time points t₀ and t₂₄ hrs, after incubation in a 37° C. tissueculture incubator. Peptide plasma half-life in minutes was estimated bycalculating the following:

1) LN((EC₅₀ at t₀)/(EC₅₀ at t_(24 hrs))),

2) Calculate slope of value above and

3) t_(1/2)=0.693/(slopê2).

Using the test assay (as described above) polypeptides of the inventionexhibited efficacy and stability in accordance to Tables 2 and 3,provided infra.

TABLE 2 Activity and Stability of Polypeptides Surrogate hAPJ Ca²⁺activity-based Flux EC₅₀ Plasma stability Peptide [nM] t½ [min] Example1 90.13 30.7 Example 2 132.58 27.1 Example 3 1008.19 98.0 Example 4746.74 50.0 Example 5 21.25 126.2 Example 6 21.98 117.2 Example 790.85 >1000 Example 8 1.04 407 Example 9 2.16 >1000 Example 102.54 >1000 Example 11 3.52 >1000 Example 12 2.07 93.6 Example 13 2.26283.6 Example 14 8.83 85.2 Example 15 3.53 180.3 Example 16 1.43 13.4Example 17 3.29 14.1 Example 18 1.62 248.1 Example 19 8.46 28.4 Example20 173.24 490.4 Example 21 75.81 639.7 Example 22 42.03 799.9 Example 2352.42 >1000 Example 24 32.65 303.5 Example 25 24.50 >1000 Example 2629.84 >1000 Example 27 65.55 >1000 Example 28 5.68 440.3 Example 29 4.33216.6 Example 30 6.05 >1000 Example 31 84.11 >1000 Example 32518.08 >1000 Example 33 8.10 >1000 Example 34 3.21 505.2 Example 3511.12 >1000 Example 36 7.36 >1000 Example 37 1.40 164.3 Example 38181.77 >1000 Example 39 8.20 654.9 Example 40 7.99 >1000 Example 41 6.91627.1 Example 42 92.77 >1000 Example 43 4.14 >1000 Example 44 5.94 855.8Example 45 6.55 167.3 Example 46 3.87 502.1 Example 47 2.41 418.9Example 48 4.10 546.4 Example 49 34.19 >1000 Example 50 1.92 >1000Example 51 1.09 655.9 Example 52 53.88 777.8 Example 53 1.07 >1000Example 54 6.70 >1000 Example 55 8.16 >1000 Example 56 1.12 >1000Example 57 2.01 >1000 Example 58 13.09 >1000 Example 59 10.00 >1000Example 60 8.15 >1000 Example 61 89.15 259.9 Example 62 3.32 >1000Example 63 3.18 703.9 Example 64 6.3 455 Example 65 1.1 596 Example 66589 596 Comparative 1.79 5.0 Example: Pyr1-apelin-13

TABLE 3 Correlation bewteen plasma stabililty Assay and SurrogateActivity based Plasma Stability assay: Surrogate Plasma Activity basedstability Plasma stability Peptide t½ [min] t½ [min] Example 8 163 407Example 12 53.9 96.16 Example 13 183 283.6 Example 14 63 85.2 Example 1610.2 13.4 Example 17 2.3 14.1 Example 18 220 248.1 Pyr-1-Apelin 13 6.65.0

The polypeptide of the present invention may have an APJ receptorpotency similar to apelin-13 or pyr-1-apelin-13. In one embodiment thepolypeptide of the present invention has an EC₅₀ of less than 100 nM. Inanother embodiment the polypeptide of the invention has an EC₅₀ of lessthan 50 nM, preferably less than 25 nM and more preferably less than 15nM. In yet another embodiment, the polypeptide of the present inventionhas an EC₅₀ of less than 10 nM.

The polypeptide of the present invention may have plasma stabilitysuperior to apelin-13 or pyr-1-apelin-13. In one embodiment, the plasmastability improvement is at least 2 fold. In one embodiment, thepolypeptide of the invention has a plasma stability of at least 30minutes. In another embodiment, the polypeptide of the invention has aplasma stability of at least 10 minutes, at least 40 min and morepreferably at least 60 minutes.

The polypeptide of the present invention may be administered eithersimultaneously with, or before or after, one or more other therapeuticagent. The polypeptide of the present invention may be administeredseparately, by the same or different route of administration, ortogether in the same pharmaceutical composition as the other agents.

In one embodiment, the invention provides a product comprising apolypeptide of anyone of formulae I to IX, or an amide, an ester of asalt thereof, and at least one other therapeutic agent as a combinedpreparation for simultaneous, separate or sequential use in therapy. Inone embodiment, the therapy is the treatment of a disease or conditionresponsive to the activation of the APJ receptor.

Products provided as a combined preparation include a compositioncomprising a polypeptide of anyone of formulae I to IX, or an amide, anester of a salt thereof, and the other therapeutic agent(s) together inthe same pharmaceutical composition, or a polypeptide of anyone offormulae I to IX, or an amide, an ester or a salt thereof, and the othertherapeutic agent(s) in separate form, e.g. in the form of a kit.

In one embodiment, the invention provides a pharmaceutical compositioncomprising a polypeptide of anyone of formulae I to IX, or an amide, anester or a salt thereof, and another therapeutic agent(s). Optionally,the pharmaceutical composition may comprise a pharmaceuticallyacceptable excipient, as described above.

In one embodiment, the invention provides a kit comprising two or moreseparate pharmaceutical compositions, at least one of which contains apolypeptide of anyone of formula I′ and I to IX, or an amide, an esteror a salt thereof. In one embodiment, the kit comprises means forseparately retaining said compositions, such as a container, dividedbottle, or divided foil packet. An example of such a kit is a blisterpack, as typically used for the packaging of tablets, capsules and thelike.

The kit of the invention may be used for administering different dosageforms, for example, oral and parenteral, for administering the separatecompositions at different dosage intervals, or for titrating theseparate compositions against one another. To assist compliance, the kitof the invention typically comprises directions for administration.

In the combination therapies of the invention, the compound of theinvention and the other therapeutic agent may be manufactured and/orformulated by the same or different manufacturers. Moreover, thecompound of the invention and the other therapeutic may be broughttogether into a combination therapy: (i) prior to release of thecombination product to physicians (e.g. in the case of a kit comprisingthe compound of the invention and the other therapeutic agent); (ii) bythe physician themselves (or under the guidance of the physician)shortly before administration; (ii) in the patient themselves, e.g.during sequential administration of a polypeptide of the invention andthe other therapeutic agent.

Accordingly, the invention provides the use of a polypeptide of anyoneof formulae I to IX, or an amide, an ester or a salt thereof, fortreating a disease or condition responsive to the agonism of the APJreceptor, wherein the medicament is prepared for administration withanother therapeutic agent. The invention also provides the use ofanother therapeutic agent for treating a disease or condition responsiveto the agonism of the apelin receptor, wherein the medicament isadministered with a polypeptide of anyone of formulae I to IX, or anamide, an ester or a salt thereof.

The invention also provides a polypeptide of anyone of formulae I to IX,or a pharmaceutically acceptable salt thereof, for use in a method oftreating a disease or condition responsive to the agonism of the APJreceptor, wherein the polypeptide of anyone of formulae I to IX, or anamide, an ester or a salt thereof, is prepared for administration withanother therapeutic agent. The invention also provides anothertherapeutic agent for use in a method of treating a disease or conditionresponsive to the agonism of the APJ receptor, wherein the othertherapeutic agent is prepared for administration with a polypeptide ofanyone of formulae I to IX, or an amide, an ester or a salt thereof. Theinvention also provides a polypeptide of anyone of formulae I to IX, oran amide, an ester or a salt thereof, for use in a method of treating adisease or condition responsive to the agonism of the APJ receptor,wherein the polypeptide of anyone of formulae I to IX, or an amide, anester or a salt thereof, is administered with another therapeutic agent.The invention also provides another therapeutic agent for use in amethod of treating a disease or condition responsive to the agonism ofthe APJ receptor, wherein the other therapeutic agent is administeredwith a polypeptide of anyone of formulae I to IX or an amide, an esteror a salt thereof.

The invention also provides the use of a polypeptide of anyone offormulae I to IX, or an amide, an ester or a salt thereof, for treatinga disease or condition responsive to the agonism of the APJ receptor,wherein the patient has previously (e.g. within 24 hours) been treatedwith another therapeutic agent. The invention also provides the use ofanother therapeutic agent for treating a disease or condition responsiveto the agonism of the APJ receptor, wherein the patient has previously(e.g. within 24 hours) been treated with a polypeptide of anyone offormulae I to IX, or an amide, an ester or a salt thereof.

In one embodiment, the other therapeutic agent is selected frominotropes, beta adrenergic receptor blockers, HMG-Co-A reductaseinhibitors, angiotensin II receptor antagonists, angiotensin convertingenzyme (ACE) Inhibitors, calcium channel blockers (CCB), endothelinantagonists, renin inhibitors, diuretics, ApoA-I mimics, anti-diabeticagents, obesity-reducing agents, aldosterone receptor blockers,endothelin receptor blockers, aldosterone synthase inhibitors (ASI), aCETP inhibitor, anti-coagulants, relaxin, BNP (nesiritide) and a NEPinhibitor.

The term “in combination with” a second agent or treatment includesco-administration of the polypeptide of the invention (e.g., apolypeptide according to anyone of Formulae I-IX or a polypeptideotherwise described herein) with the second agent or treatment,administration of the compound of the invention first, followed by thesecond agent or treatment and administration of the second agent ortreatment first, followed by the compound of the invention.

The term “second agent” includes any agent which is known in the art totreat, prevent, or reduce the symptoms of a disease or disorderdescribed herein, e.g. a disorder or disease responsive to theactivation of the APJ receptor, such as for example, acute decompensatedheart failure (ADHF), chronic heart failure, pulmonary hypertension,atrial fibrillation, Brugada syndrome, ventricular tachycardia,atherosclerosis, hypertension, restenosis, ischemic cardiovasculardiseases, cardiomyopathy, cardiac fibrosis, arrhythmia, water retention,diabetes (including gestational diabetes), obesity, peripheral arterialdisease, cerebrovascular accidents, transient ischemic attacks,traumatic brain injuries, amyotrophic lateral sclerosis, burn injuries(including sunburn) and preeclampsia.

Examples of second agents include inotropes, beta adrenergic receptorblockers, HMG-Co-A reductase inhibitors, angiotensin II receptorantagonists, angiotensin converting enzyme (ACE) Inhibitors, calciumchannel blockers (CCB), endothelin antagonists, renin inhibitors,diuretics, ApoA-I mimics, anti-diabetic agents, obesity-reducing agents,aldosterone receptor blockers, endothelin receptor blockers, aldosteronesynthase inhibitors (ASI), a CETP inhibitor, anti-coagulants, relaxin,BNP (nesiritide) and/or a NEP inhibitor.

Inotropes as used herein include for example dobutamine, isoproterenol,milrinone, amirinone, levosimendan, epinephrine, norepinephrine,isoproterenol and digoxin.

Beta adrenergic receptor blockers as used herein include for exampleacebutolol, atenolol, betaxolol, bisoprolol, carteolol, metoprolol,nadolol, propranolol, sotalol and timolol.

Anti-coagulants as used herein include Dalteparin, Danaparoid,Enoxaparin, Heparin, Tinzaparin, Warfarin.

The term “HMG-Co-A reductase inhibitor” (also calledbeta-hydroxy-beta-methylglutaryl-co-enzyme-A reductase inhibitors)includes active agents that may be used to lower the lipid levelsincluding cholesterol in blood. Examples include atorvastatin,cerivastatin, compactin, dalvastatin, dihydrocompactin, fluindostatin,fluvastatin, lovastatin, pitavastatin, mevastatin, pravastatin,rosuvastatin, rivastatin, simvastatin, and velostatin, or,pharmaceutically acceptable salts thereof.

The term “ACE-inhibitor” (also called angiotensin converting enzymeinhibitors) includes molecules that interrupt the enzymatic degradationof angiotensin I to angiotensin II. Such compounds may be used for theregulation of blood pressure and for the treatment of congestive heartfailure. Examples include alacepril, benazepril, benazeprilat,captopril, ceronapril, cilazapril, delapril, enalapril, enaprilat,fosinopril, imidapril, lisinopril, moexipril, moveltopril, perindopril,quinapril, ramipril, spirapril, temocapril, and trandolapril, or,pharmaceutically acceptables salt thereof.

The term “endothelin antagonist” includes bosentan (cf. EP 526708 A),tezosentan (cf. WO 96/19459), or, pharmaceutically acceptable saltsthereof.

The term “renin inhibitor” includes ditekiren (chemical name:[1S-[1R*,2R*,4R*(1R*,2R*)]]-1-[(1,1-dimethylethoxy)carbonyl]-L-prolyI-L-phenylalanyl-N-[2-hydroxy-5-methyl-1-(2-methylpropyl)-4-[[[2-methyl-1-[[(2-pyridinylmrthyl)amino]carbonyl]butyl]amino]carbonyl]hexyl]-N-alfa-methyl-L-histidinamide);terlakiren (chemical name:[R—(R*,S*)]-N-(4-morpholinylcarbonyl)-L-phenylalanyl-N-[1-(cyclohexylmethyl)-2-hydroxy-3-(1-methylethoxy)-3-oxopropyl]-S-methyl-L-cysteineamide);Aliskiren (chemical name:(2S,4S,5S,7S)-5-amino-N-(2-carbamoyl-2,2-dimethylethyl)-4-hydroxy-7-{[4-methoxy-3-(3-methoxypropoxy)phenyl]methyl}-8-methyl-2-(propan-2-yl)nonanamide) and zankiren(chemical name:[1S-[1R*[R*(R*)],2S*,3R]]-N-[1-(cyclohexylmethyl)-2,3-dihydroxy-5-methylhexyl]-alfa-[[2-[[(4-methyl-1-piperazinyl)sulfonyl]methyl]-1-oxo-3-phenylpropyl]-amino]-4-thiazolepropanamide),or, hydrochloride salts thereof, or, SPP630, SPP635 and SPP800 asdeveloped by Speedel, or RO 66-1132 and RO 66-1168 of Formula (A) and(B):

or, pharmaceutically acceptable salts thereof.

The term “aliskiren”, if not defined specifically, is to be understoodboth as the free base and as a salt thereof, especially apharmaceutically acceptable salt thereof, most preferably ahemi-fumarate salt thereof.

The term “calcium channel blocker (CCB)” includes dihydropyridines(DHPs) and non-DHPs (e.g., diltiazem-type and verapamil-type CCBs).Examples include amlodipine, Bepridil, Diltiazem, felodipine, ryosidine,isradipine, lacidipine, nicardipine, nifedipine, niguldipine,niludipine, nimodipine, nisoldipine, nitrendipine, Verapamil andnivaldipine, and is preferably a non-DHP representative selected fromthe group consisting of flunarizine, prenylamine, diltiazem, fendiline,gallopamil, mibefradil, anipamil, tiapamil and verapamil, or,pharmaceutically acceptable salts thereof. CCBs may be used asanti-hypertensive, anti-angina pectoris, or anti-arrhythmic drugs.

The term “diuretic” includes thiazide derivatives (e.g., chlorothiazide,hydrochlorothiazide, methylclothiazide, and chlorothalidon).

The term “ApoA-I mimic” includes D4F peptides (e.g., formulaD-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F)

An angiotensin II receptor antagonist or a pharmaceutically acceptablesalt thereof is understood to be an active ingredient which bind to theAT₁-receptor subtype of angiotensin II receptor but do not result inactivation of the receptor. As a consequence of the inhibition of theAT₁ receptor, these antagonists can, for example, be employed asantihypertensives or for treating congestive heart failure.

The class of AT₁ receptor antagonists comprises compounds havingdiffering structural features, essentially preferred are thenon-peptidic ones. For example, mention may be made of the compoundswhich are selected from the group consisting of valsartan, losartan,candesartan, eprosartan, irbesartan, saprisartan, tasosartan,telmisartan, the compound with the designation E-1477 of the followingformula

the compound with the designation SC-52458 of the following formula

and the compound with the designation ZD-8731 of the following formula

or, in each case, a pharmaceutically acceptable salt thereof.

Preferred AT₁-receptor antagonist are candesartan, eprosartan,irbesartan, losartan, telmisartan, valsartan. Also preferred are thoseagents which have been marketed, most preferred is valsartan or apharmaceutically acceptable salt thereof.

The term “anti-diabetic agent” includes insulin secretion enhancers thatpromote the secretion of insulin from pancreatic β-cells. Examplesinclude biguanide derivatives (e.g., metformin), sulfonylureas (SU)(e.g., tolbutamide, chlorpropamide, tolazamide, acetohexamide,4-chloro-N-[(1-pyrolidinylamino)carbonyl]-benzensulfonamide(glycopyramide), glibenclamide (glyburide), gliclazide,1-butyl-3-nnetanilylurea, carbutamide, glibonuride, glipizide,gliquidone, glisoxepid, glybuthiazole, glibuzole, glyhexamide,glymidine, glypinamide, phenbutamide, and tolylcyclamide), orpharmaceutically acceptable salts thereof. Further examples includephenylalanine derivatives (e.g., nateglinide[N-(trans-4-isopropylcyclohexylcarbonyl)-D-phenylalanine] (cf. EP 196222and EP 526171) of the formula

repaglinide[(S)-2-ethoxy-4-{2-[[3-methyl-1-[2-(1-piperidinyl)phenyl]butyl]amino]-2-oxoethyl}benzoicacid] (cf. EP 589874, EP 147850 A2, in particular Example 11 on page 61,and EP 207331 A1); calcium(2S)-2-benzyl-3-(cis-hexahydro-2-isoindolinlycarbonyl)-propionatedihydrate (e.g., mitiglinide (cf. EP 507534)); and glimepiride (cf. EP31058).

Further examples of second agents with which the peptide and polypeptideof the invention can be used in combination include DPP-IV inhibitors,GLP-1 and GLP-1 agonists.

DPP-IV is responsible for inactivating GLP-1. More particularly, DPP-IVgenerates a GLP-1 receptor antagonist and thereby shortens thephysiological response to GLP-1. GLP-1 is a major stimulator ofpancreatic insulin secretion and has direct beneficial effects onglucose disposal.

The DPP-IV (dipeptidyl peptidase IV) inhibitor can be peptidic or,preferably, non-peptidic. DPP-IV inhibitors are in each case genericallyand specifically disclosed e.g. in WO 98/19998, DE 196 16 486 A1, WO00/34241 and WO 95/15309, in each case in particular in the compoundclaims and the final products of the working examples, thesubject-matter of the final products, the pharmaceutical preparationsand the claims are hereby incorporated into the present application byreference to these publications. Preferred are those compounds that arespecifically disclosed in Example 3 of WO 98/19998 and Example 1 of WO00/34241, respectively.

GLP-1 (glucagon like peptide-1) is an insulinotropic protein which isdescribed, e.g., by W.E. Schmidt et al. in Diabetologia, 28, 1985,704-707 and in U.S. Pat. No. 5,705,483.

The term “GLP-1 agonists” includes variants and analogs ofGLP-1(7-36)NH₂ which are disclosed in particular in U.S. Pat. No.5,120,712, U.S. Pat. No. 5,118,666, U.S. Pat. No. 5,512,549, WO 91/11457and by C. Orskov et al in J. Biol. Chem. 264 (1989) 12826. Furtherexamples include GLP-1(7-37), in which compound the carboxy-terminalamide functionality of Arg³⁶ is displaced with Gly at the 37^(th)position of the GLP-1(7-36)NH₂ molecule and variants and analogs thereofincluding GLN⁹-GLP-1(7-37), D-GLN⁹-GLP-1(7-37), acetyl LYS⁹-GLP-1(7-37),LYS¹⁸-GLP-1(7-37) and, in particular, GLP-1(7-37)OH, VAL⁸-GLP-1(7-37),GLY⁸-GLP-1(7-37), THR⁸-GLP-1(7-37), MET⁸-GLP-1(7-37) and4-imidazopropionyl-GLP-1. Special preference is also given to the GLPagonist analog exendin-4, described by Greig et al. in Diabetologia1999, 42, 45-50.

Also included in the definition “anti-diabetic agent” are insulinsensitivity enhancers which restore impaired insulin receptor functionto reduce insulin resistance and consequently enhance the insulinsensitivity. Examples include hypoglycemic thiazolidinedione derivatives(e.g., glitazone,(S)-((3,4-dihydro-2-(phenyl-methyl)-2H-1-benzopyran-6-yl)methyl-thiazolidine-2,4-dione(englitazone),5-{[4-(3-(5-methyl-2-phenyl-4-oxazolyl)-1-oxopropyl)-phenyl]methyl}-thiazolidine-2,4-dione(darglitazone),5-{[4-(1-methyl-cyclohexyl)methoxy)-phenyl]methyl}thiazolidine-2,4-dione(ciglitazone),5-{[4-(2-(1-indolyl)ethoxy)phenyl]methyl}-thiazolidine-2,4-dione(DRF2189),5-{4-[2-(5-methyl-2-phenyl-4-oxazolyl)-ethoxy)benzyl}-thiazolidine-2,4-dione(BM-13.1246), 5-(2-naphthylsulfonyl)-thiazolidine-2,4-dione (AY-31637),bis{4-[(2,4-dioxo-5-thiazolidinyl)methyl]phenyl}methane (YM268),5-{4-[2-(5-methyl-2-phenyl-4-oxazolyl)-2-hydroxyethoxy]benzyl}-thiazolidine-2,4-dione(AD-5075),5-[4-(1-phenyl-1-cyclopropanecarbonylamino)-benzyl]-thiazolidine-2,4-dione(DN-108)5-{[4-(2-(2,3-dihydroindol-1-yl)ethoxy)phenyl]methyl}-thiazolidine-2,4-dione,5-[3-(4-chloro-phenyl])-2-propynyl]-5-phenylsulfonyl)thiazolidine-2,4-dione,5-[3-(4-chlorophenyl])-2-propynyl]-5-(4-fluorophenyl-sulfonyl)thiazolidine-2,4-dione,5-{[4-(2-(methyl-2-pyridinyl-amino)-ethoxy)phenyl]methyl}-thiazolidine-2,4-dione(rosiglitazone),5-{[4-(2-(5-ethyl-2-pyridyl)ethoxy)phenyl]-methyl}thiazolidine-2,4-dione(pioglitazone),5-{[4-((3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)methoxy)-phenyl]-methyl}-thiazolidine-2,4-dione(troglitazone),5-[6-(2-fluoro-benzyloxy)naphthalen-2-ylmethyl]-thiazolidine-2,4-dione(MCC555),5-{[2-(2-naphthyl)-benzoxazol-5-yl]-methyl}thiazolidine-2,4-dione(T-174) and5-(2,4-dioxothiazolidin-5-ylmethyl)-2-methoxy-N-(4-trifluoromethyl-benzyl)benzamide(KRP297)).

Further anti-diabetic agents include, insulin signalling pathwaymodulators, like inhibitors of protein tyrosine phosphatases (PTPases),antidiabetic non-small molecule mimetic compounds and inhibitors ofglutamine-fructose-6-phosphate amidotransferase (GFAT); compoundsinfluencing a dysregulated hepatic glucose production, like inhibitorsof glucose-6-phosphatase (G6Pase), inhibitors offructose-1,6-bisphosphatase (F-1,6-Bpase), inhibitors of glycogenphosphorylase (GP), glucagon receptor antagonists and inhibitors ofphosphoenolpyruvate carboxykinase (PEPCK); pyruvate dehydrogenase kinase(PDHK) inhibitors; inhibitors of gastric emptying; insulin; inhibitorsof GSK-3; retinoid X receptor (RXR) agonists; agonists of Beta-3 AR;agonists of uncoupling proteins (UCPs); non-glitazone type PPARγagonists; dual PPARα/PPARγ agonists; antidiabetic vanadium containingcompounds; incretin hormones, like glucagon-like peptide-1 (GLP-1) andGLP-1 agonists; beta-cell imidazoline receptor antagonists; miglitol;α₂-adrenergic antagonists; and pharmaceutically acceptable saltsthereof.

In one embodiment, the invention provides a combination, in particular apharmaceutical combination, comprising a therapeutically effectiveamount of the polypeptide according to the definition of anyone offormulae I to IX, or an amide, an ester or a salt thereof, and one ormore therapeutically active agents selected from β-adrenergic receptorblockers such as acebutolol, atenolol, betaxolol, bisoprolol,metoprolol, nadolol, propranolol, sotalol and timolol; angiotensin IIreceptor antagonists such as AT1 blockers; antidiabetic agents such asDPPIV inhibitors (e.g. vildagliptin) and GLP1 peptide agonist.

The term “obesity-reducing agent” includes lipase inhibitors (e.g.,orlistat) and appetite suppressants (e.g., sibutramine and phentermine).

An aldosterone synthase inhibitor or a pharmaceutically acceptable saltthereof is understood to be an active ingredient that has the propertyto inhibit the production of aldosterone. Aldosterone synthase (CYP11B2)is a mitochondrial cytochrome P450 enzyme catalyzing the last step ofaldosterone production in the adrenal cortex, i.e., the conversion of11-deoxycorticosterone to aldosterone. The inhibition of the aldosteroneproduction with so-called aldosterone synthase inhibitors is known to bea successful variant to treatment of hypokalemia, hypertension,congestive heart failure, atrial fibrillation or renal failure. Suchaldosterone synthase inhibition activity is readily determined by thoseskilled in the art according to standard assays (e.g., US 2007/0049616).

The class of aldosterone synthase inhibitors comprises both steroidaland non-steroidal aldosterone synthase inhibitors, the later being mostpreferred.

Preference is given to commercially available aldosterone synthaseinhibitors or those aldosterone synthase inhibitors that have beenapproved by the health authorities.

The class of aldosterone synthase inhibitors comprises compounds havingdiffering structural features. An example of non-steroidal aldosteronesynthase inhibitor is the (+)-enantiomer of the hydrochloride offadrozole (U.S. Pat. Nos. 4,617,307 and 4,889,861) of formula

or, if appropriable, a pharmaceutically acceptable salt thereof.

Aldosterone synthase inhibitors useful in said combination are compoundsand analogs generically and specifically disclosed e.g. inUS2007/0049616, in particular in the compound claims and the finalproducts of the working examples, the subject-matter of the finalproducts, the pharmaceutical preparations and the claims are herebyincorporated into the present application by reference to thispublication. Preferred aldosterone synthase inhibitors suitable for usein the present invention include, without limitation4-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-3-methylbenzonitrile;5-(2-chloro-4-cyanophenyl)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazole-5-carboxylicacid (4-methoxybenzyl)methylamide;4′-fluoro-6-(6,7,8,9-tetrahydro-5H-imidazo[1,5-a]azepin-5-yl)biphenyl-3-carbonitrile;5-(4-Cyano-2-methoxyphenyl)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazole-5-carboxylicacid butyl ester;4-(6,7-Dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-2-methoxybenzonitrile;5-(2-Chloro-4-cyanophenyl)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazole-5-carboxylicacid 4-fluorobenzyl ester;5-(4-Cyano-2-trifluoromethoxyphenyl)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazole-5-carboxylicacid methyl ester;5-(4-Cyano-2-methoxyphenyl)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazole-5-carboxylicacid 2-isopropoxyethyl ester;4-(6,7-Dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-2-methylbenzonitrile;4-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-3-fluorobenzonitrile;4-(6,7-Dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-2-methoxybenzonitrile;3-Fluoro-4-(7-methylene-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)benzonitrile;cis-3-Fluoro-4-[7-(4-fluoro-benzyl)-5,6,7,8-tetrahydro-imidazo[1,5-a]pyridin-5-yl]benzonitrile;4′-Fluoro-6-(9-methyl-6,7,8,9-tetrahydro-5H-imidazo[1,5-a]azepin-5-yl)biphenyl-3-carbonitrile;4′-Fluoro-6-(9-methyl-6,7,8,9-tetrahydro-5H-imidazo[1,5-a]azepin-5-yl)biphenyl-3-carbonitrileor in each case, the (R) or (S) enantiomer thereof; or if appropriable,a pharmaceutically acceptable salt thereof.

The term aldosterone synthase inhibitors also include compounds andanalogs disclosed in WO2008/076860, WO2008/076336, WO2008/076862,WO2008/027284, WO2004/046145, WO2004/014914, WO2001/076574.

Furthermore Aldosterone synthase inhibitors also include compounds andanalogs disclosed in U.S. patent applications US2007/0225232,US2007/0208035, US2008/0318978, US2008/0076794, US2009/0012068,US20090048241 and in PCT applications WO2006/005726, WO2006/128853,WO2006128851, WO2006/128852, WO2007065942, WO2007/116099, WO2007/116908,WO2008/119744 and in European patent application EP 1886695. Preferredaldosterone synthase inhibitors suitable for use in the presentinvention include, without limitation8-(4-Fluorophenyl)-5,6-dihydro-8H-imidazo[5,1-c1[1,41oxazine;4-(5,6-Dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)-2-fluorobenzonitrile;4-(5,6-Dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)-2,6-difluorobenzonitrile;4-(5,6-Dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)-2-methoxybenzonitrile;3-(5,6-Dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)benzonitrile;4-(5,6-Dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)phthalonitrile;4-(8-(4-Cyanophenyl)-5,6-dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)benzonitrile;4-(5,6-Dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)benzonitrile;4-(5,6-Dihydro-8H-imidazo[5,1-c][1,4]oxazin-8-yl)naphthalene-1-carbonitrile;8-[4-(1H-Tetrazol-5-yl)phenyl1-5,6-dihydro-8H-imidazo[5,1-c][1,4]oxazineas developed by Speedel or in each case, the (R) or (S) enantiomerthereof; or if appropriable, a pharmaceutically acceptable salt thereof.

Aldosterone synthase inhibitors useful in said combination are compoundsand analogs generically and specifically disclosed e.g. in WO2009/156462 and WO 2010/130796, in particular in the compound claims andthe final products of the working examples, the subject-matter of thefinal products, the pharmaceutical preparations and the claims.Preferred Aldosterone Synthase inhibitors suitable for combination inthe present invention include,3-(6-Fluoro-3-methyl-2-pyridin-3-yl-1H-indol-1-ylmethyl)-benzonitrilehydrochloride,1-(4-Methanesulfonyl-benzyl)-3-methyl-2-pyridin-3-yl-1H-indole,2-(5-Benzyloxy-pyridin-3-yl)-6-chloro-1-methyl-1H-indole,5-(3-Cyano-1-methyl-1H-indol-2-yl)-nicotinic acid ethyl ester,N-[5-(6-chloro-3-cyano-1-methyl-1H-indol-2-yl)-pyridin-3-ylmethyl]-ethanesulfonamide,Pyrrolidine-1-sulfonic acid5-(6-chloro-3-cyano-1-methyl-1H-indol-2-yl)-pyridin-3-yl ester,N-Methyl-N-[5-(1-methyl-1H-indol-2-yl)-pyridin-3-ylmethyl]-methanesulfonamide,6-Chloro-1-methyl-2-{5-[(2-pyrrolidin-1-yl-ethylamino)-methyl]-pyridin-3-yl}-1H-indole-3-carbonitrile,6-Chloro-2-[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-pyridin-3-yl]-1-methyl-1H-indole-3-carbonitrile,6-Chloro-1-methyl-2-{5-[(1-methyl-piperidin-4-ylamino)-methyl]-pyridin-3-yl}-1H-indole-3-carbonitrile,Morpholine-4-carboxylic acid[5-(6-chloro-3-cyano-1-methyl-1H-indol-2-yl)-pyridin-3-ylmethyl]-amide,N-[5-(6-Chloro-1-methyl-1H-indol-2-yl)-pyridin-3-ylmethyl]-ethanesulfonamide,C,C,C-Trifluoro-N-[5-(1-methyl-1H-indol-2-yl)-pyridin-3-ylmethyl]methanesulfonamide,N-[5-(3-Chloro-4-cyano-phenyl)-pyridin-3-yl]-4-trifluoromethyl-benzenesulfonamide,N-[5-(3-Chloro-4-cyano-phenyl)-pyridin-3-yl]-1-phenyl-methanesulfonamide,N-(5-(3-chloro-4-cyanophenyl)pyridin-3-yl)butane-1-sulfonamide,N-(1-(5-(4-cyano-3-methoxyphenyl)pyridin-3-yl)ethyl)ethanesulfonamide,N-((5-(3-chloro-4-cyanophenyl)pyridin-3-yl)(cyclopropyl)methyl)ethanesulfonamide,N-(cyclopropyl(5-(1H-indol-5-yl)pyridin-3-yl)methyl)ethanesulfonamide,N-(cyclopropyl(5-naphtalen-1-yl-pyridin-3-yl)methyl)ethanesulfonamide,Ethanesulfonic acid[5-(6-chloro-1-methyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-pyridin-3-ylmethyl]-amideand Ethanesulfonic acid{[5-(3-chloro-4-cyano-phenyl)-pyridin-3-yl]-cyclopropyl-methyl}-ethyl-amide.

The term “endothelin receptor blocker” includes bosentan andambrisentan.

The term “CETP inhibitor” refers to a compound that inhibits thecholesteryl ester transfer protein (CETP) mediated transport of variouscholesteryl esters and triglycerides from HDL to LDL and VLDL. Such CETPinhibition activity is readily determined by those skilled in the artaccording to standard assays (e.g., U.S. Pat. No. 6,140,343). Examplesinclude compounds disclosed in U.S. Pat. No. 6,140,343 and U.S. Pat. No.6,197,786 (e.g.,[2R,4S]4-[(3,5-bis-trifluoromethyl-benzyl)-methoxycarbonyl-amino]-2-ethyl-6-trifluoromethyl-3,4-dihydro-2H-quinoline-1-carboxylicacid ethyl ester (torcetrapib); compounds disclosed in U.S. Pat. No.6,723,752 (e.g.,(2R)-3-{[3-(4-Chloro-3-ethyl-phenoxy)-phenyl]-[[3-(1,1,2,2-tetrafluoro-ethoxy)-phenyl]-methyl]-amino}-1,1,1-trifluoro-2-propanol);compounds disclosed in U.S. patent application Ser. No. 10/807,838;polypeptide derivatives disclosed in U.S. Pat. No. 5,512,548;rosenonolactone derivatives and phosphate-containing analogs ofcholesteryl ester disclosed in J. Antibiot, 49(8): 815-816 (1996), andBioorg. Med Chem. Lett.; 6:1951-1954 (1996), respectively. Furthermore,the CETP inhibitors also include those disclosed in WO2000/017165,WO2005/095409, WO2005/097806, WO 2007/128568, WO2008/009435, WO2009/059943 and WO2009/071509.

The term “NEP inhibitor” refers to a compound that inhibits neutralendopeptidase (NEP) EC 3.4.24.11. Examples include Candoxatril,Candoxatrilat, Dexecadotril, Ecadotril, Racecadotril, Sampatrilat,Fasidotril, Omapatrilat, Gemopatrilat, Daglutril, SCH-42495, SCH-32615,UK-447841, AVE-0848, PL-37 and and(2R,4S)-5-Biphenyl-4-yl-4-(3-carboxy-propionylamino)-2-methyl-pentanoicacid ethyl ester or a pharmaceutically acceptable salt thereof. NEPinhibitors also include Phosphono/biaryl substituted dipeptidederivatives, as disclosed in U.S. Pat. No. 5,155,100. NEP inhibitorsalso include N-mercaptoacyl phenylalanine derivative as disclosed in PCTapplication Number WO 2003/104200. NEP inhibitors also includedual-acting antihypertensive agents as disclosed in PCT applicationNumbers WO 2008/133896, WO 2009/035543 or WO 2009/134741. Other examplesinclude compounds disclosed in U.S. application Ser. Nos. 12/788,794;12/788,766 and 12/947,029. NEP inhibitors also include compoundsdisclosed in WO 2010/136474, WO 2010/136493, WO 2011/061271 and U.S.provisional applications No 61/414,171 and 61/414,163.

In one embodiment, the invention provides a method of activating the APJreceptor in a subject, wherein the method comprises administering to thesubject a therapeutically effective amount of the polypeptide accordingto the definition of anyone of formulae I to IX, or an amide, an esteror a salt thereof.

In one embodiment, the invention provides a method of treating adisorder or a disease responsive to the activation of the APJ receptor,in a subject, wherein the method comprises administering to the subjecta therapeutically effective amount of the polypeptide according to thedefinition of anyone of formulae I to IX, or an amide, an ester or asalt thereof.

In one embodiment, the invention provides a method of treating adisorder or a disease responsive to the activation (agonism) of the APJreceptor, in a subject, wherein the disorder or the disease is selectedfrom acute decompensated heart failure (ADHF), chronic heart failure,pulmonary hypertension, atrial fibrillation, Brugada syndrome,ventricular tachycardia, atherosclerosis, hypertension, restenosis,ischemic cardiovascular diseases, cardiomyopathy, cardiac fibrosis,arrhythmia, water retention, diabetes (including gestational diabetes),obesity, peripheral arterial disease, cerebrovascular accidents,transient ischemic attacks, traumatic brain injuries, amyotrophiclateral sclerosis, burn injuries (including sunburn) and preeclampsia.

In one embodiment, the invention provides a polypeptide according to thedefinition of anyone of formulae I to IX, for use as a medicament.

In one embodiment, the invention provides the use of a polypeptideaccording to the definition of anyone of formulae I to IX, or an amide,an ester or a salt thereof, in the manufacture of a medicament, for thetreatment of a disorder or disease responsive to the activation of theAPJ receptor. In another embodiment, the invention provides the use of apolypeptide according to the definition of anyone of formulae I to IX,or an amide, an ester or a salt thereof, in the manufacture of amedicament, for the treatment of a disorder or disease responsive to theactivation of the APJ receptor, wherein said disorder or disease is inparticular selected from acute decompensated heart failure (ADHF),chronic heart failure, pulmonary hypertension, atrial fibrillation,Brugada syndrome, ventricular tachycardia, atherosclerosis,hypertension, restenosis, ischemic cardiovascular diseases,cardiomyopathy, cardiac fibrosis, arrhythmia, water retention, diabetes(including gestational diabetes), obesity, peripheral arterial disease,cerebrovascular accidents, transient ischemic attacks, traumatic braininjuries, amyotrophic lateral sclerosis, burn injuries (includingsunburn) and preeclampsia.

Exemplification of the Invention: Peptide and Polypeptide Synthesis

Abbreviation Definition AA Amino acid Ac Acetyl Acm Acetamidomethyl ACNAcetonitrile AcOH Acetic acid Ac₂O Acetic anhydride AM Aminomethyl BALBackbone amide linker BSA Bovine Serum Albumin Boc tert-ButyloxycarbonylDCM Dichlormethane DIC N,N′-Diisopropylcarbodiimide DIPEAN,N′-Diisopropylethylamine DMA N,N′-Dimethylacetamide DMFN,N′-Dimethylformamide DMSO Dimethylsulfoxide DVB Divinylbenzene EDTEthanedithiol FA Formic acid Fmoc 9-Fluorenylmethyloxycarbonyl HATU2-(1H-9-Azabenzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate HBSS Hank's buffered salt solution HCTU2-(6-Chloro-1H-Benzotriazole-yl)-1,1,3,3- tetramethyluroniumhexafluorophosphate HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid HFIP Hexafluoroisopropanol HOAt 1-Hydroxy-7-azabenzotriazole HPLCHigh performance liquid chromatography ivDde(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl LN Logarithmusnaturali (natural logarithm) MeOH Methanol MS Mass spectrometry Nal2-Naphthylalanine Nle Norleucine NMP N-Methylpyrrolidine Oxyma PureEthyl 2-cyano-2-(hydroxyimino)acetate Pbf2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl pE Pyroglutamate PGProtecting group Ph Phenyl PS Polystyrene POL Polymer support rt Roomtemperature SPPS Solid phase peptide synthesis tBuOH tert-Butanol TFATrifluoroacetic acid THF Tetrahydrofuran TIS Triisopropylsilane t_(R)Retention time Trt Trityl UPLC Ultra performance liquid chromatographyUV Ultraviolet

The peptides were synthesized by standard solid phase Fmoc chemistry.The peptides were assembled on the Prelude™ peptide synthesizer (ProteinTechnologies, Inc., Tucson, USA). Peptides with a free carboxylic acidon the C-terminus were synthesized from 2-chlorotrityl chloride-PS-resin(ABCR, Karlsruhe, Germany). Peptides with an unsubstituted carboxamideon the C-terminus were synthesized from Fmoc protectedRink-Amide-AM-PS-resin (Merck, Darmstadt, Germany). Peptides with anN-monosubstituted carboxamide on the C-terminus were synthesized fromBAL-AM-PS-resin loaded with amines (EMC Microcollections, Tubingen,Germany).

The peptides were purified by preparative reversed-phase HPLC. Thefollowing columns were used:

-   -   Waters SunFire Prep C18 OBD Column, 5 μm, 30×100 mm, Part No.        186002572 (one column or two columns in series)    -   Waters SunFire Prep C18 OBD Column, 5 μm, 30×150 mm, Part No.        186002797    -   Waters Atlantis Prep OBD T3 Column, 5 μm, 30×150 mm, Part No.        186003703    -   Waters XBridge Prep C8 OBD Column, 5 μm, 30×150 mm, Part No.        186003083    -   Machery-Nagel Nucleosil® 100-5 C18, 5 μm, 250×40 mm, Part No.        715340.400

Mobile phases consisted of eluent A (0.1% TFA in H₂O) and eluent B(ACN). Gradients were designed based on the specific requirements of theseparation problem. Pure products were lyophilized from ACN/H₂O.

The products were analyzed by analytical HPLC using UV detection atλ=214 nm (Column: Bischoff UHC-640, 53×4.0 mm, ProntoSil 120-3-C18-H, 3μm, Part No. 0604F185PS030) Mobile phases consisted of eluent A (0.07%TFA in H₂O) and eluent B (0.1% TFA in ACN). Additional characterizationof the products was done by UPLC-MS (Column: Waters Acquity UPLC® BEHC18, 1.7 μm, 2.1×50 mm, Part No. 186002350) using electrosprayionization.

The peptides that are exemplified in Table 4 were synthesized using thegeneral procedures described below. Unsubstituted N- or C-termini areindicated by small italic H— or —OH, respectively.

TABLE 4 Example Sequence Type of Ring Example 1pE-R-P-R-L-K-H-F-G-P-Nle-D-Phenethylamine Lactam K⁶-D¹² Example 2pE-R-P-R-L-K-H-F-G-P-Nle-E-Phenethylamine Lactam K⁶-E¹² Example 3pE-R-P-R-L-Orn-H-F-G-P-Nle-D-Phenethylamine Lactam O⁶-D¹² Example 4pE-R-P-R-L-Dab-H-F-G-P-Nle-D-Phenethylamine Lactam Dab⁶-D¹² Example 5pE-R-P-R-L-K-F-K-G-P-Nle-F Lactam K⁶-C-terminus Example 6pE-R-P-R-L-K-F-K-G-P-Nle-f Lactam K⁶-C-terminus Example 7Q-R-P-R-L-C-F-K-G-P-Nle-C-F-G-G Lactam N-terminus-C- terminusDisulfide C⁶-C¹² Example 8 pE-R-P-R-L-C-H-K-G-P-Nle-C-F-OHDisulfide C⁶-C¹² Example 9 pE-R-P-R-L-C-Aib-K-G-P-Nle-C-F-OHDisulfide C⁶-C¹² Example 10 pE-R-P-R-L-C-Aib-K-G-P-Nle-C-f-OHDisulfide C⁶-C¹² Example 11 H-Isn-R-P-R-L-C-Aib-K-G-P-Nle-C-f-OHDisulfide C⁶-C¹² Example 12 pE-R-P-R-L-C-H-K-G-P-Nle-C-PhenethylamineDisulfide C⁶-C¹² Example 13 pE-R-P-R-L-C-H-K-G-P-Nle-C-f-OHDisulfide C⁶-C¹² Example 14 pE-R-P-R-Cha-C-H-K-G-P-Cha-C-F-OHDisulfide C⁶-C¹² Example 15 pE-R-P-R-L-C-F-K-G-P-Nle-C-F-OHDisulfide C⁶-C¹² Example 16 H-R-P-R-L-C-H-K-G-P-Nle-C-F-OHDisulfide C⁵-C¹¹ Example 17 H-R-R-P-R-L-C-H-K-G-P-Nle-C-F-OHDisulfide C⁶-C¹² Example 18 H-Isn-R-P-R-L-C-H-K-G-P-Nle-C-F-OHDisulfide C⁶-C¹² Example 19 pE-R-P-R-L-C-H-F-G-P-Nle-C-PhenethylarnineDisulfide C⁶-C¹² Example 20 pE-R-P-R-L-C-H-K-Aib-P-Nle-C-F-OHDisulfide C⁶-C¹² Example 21 pE-R-P-R-L-C-H-(4-NH-Isn)-G-P-Nle-C-F-OHDisulfide C⁶-C¹² Example 22 pE-R-P-R-L-C-H-K-G-P-Nle-C-K(PalmitoyI)-OHDisulfide C⁶-C¹² Example 23 pE-R-P-R-L-C-K(PalmitoyI)-K-G-P-Nle-C-F-OHDisulfide C⁶-C¹² Example 24Palmitoy1-020c-Q-R-P-R-L-C-H-K-G-P-Nle-C-F-OH Disulfide C⁶-C¹²Example 25 Lauroyl-020c-Q-R-P-R-L-C-H-K-G-P-Nle-C-F-OH Disulfide C⁶-C¹²Example 26 pE-R-P-R-L-C-H-K-G-P-Nle-C-K(Lauroyl)-OH Disulfide C⁶-C¹²Example 27 pE-R-P-R-L-C-K(Lau royI)-K-G-P-Nle-C-F-OH Disulfide C⁶-C¹²Example 28 pE-R-P-C-L-C-C-K-G-P-Nle-C-F-OH Disulfides C⁶-C^(12,) C⁴-C⁷Example 29 pE-R-C-R-L-C-C-K-G-P-Nle-C-F-OH Disulfides C⁶-C^(12,) C³-C⁷Example 30 pE-r-P-R-L-C-H-K-G-P-Nle-C-F-OH Disulfide C⁶-C¹² Example 31pE-F-P-R-L-C-H-K-G-P-Nle-C-F-OH Disulfide C⁶-C¹² Example 32pE-E-P-R-L-C-H-K-G-P-Nle-C-F-OH Disulfide C⁶-C¹² Example 33pE-R-p-R-L-C-H-K-G-P-Nle-C-F-OH Disulfide C⁶-C¹² Example 34pE-R-K-R-L-C-H-K-G-P-Nle-C-F-OH Disulfide C⁶-C¹² Example 35pE-R-D-R-L-C-H-K-G-P-Nle-C-F-OH Disulfide C⁶-C¹² Example 36pE-R-P-F-L-C-H-K-G-P-Nle-C-F-OH Disulfide C⁶-C¹² Example 37pE-R-P-R-K-C-H-K-G-P-Nle-C-F-OH Disulfide C⁶-C¹² Example 38pE-R-P-R-L-C-H-E-G-P-Nle-C-F-OH Disulfide C⁶-C¹² Example 39pE-R-P-R-L-C-H-K-D-P-Nle-C-F-OH Disulfide C⁶-C¹² Example 40pE-R-P-E-L-C-H-K-G-P-Nle-C-F-OH Disulfide C⁶-C¹² Example 41pE-R-P-R-(4-PhF)-C-H-K-G-P-Nle-C-F-OH Disulfide C⁶-C¹² Example 42pE-R-P-R-D-C-H-K-G-P-Nle-C-F-OH Disulfide C⁶-C¹² Example 43pE-R-P-R-L-C-E-K-G-P-Nle-C-F-OH Disulfide C⁶-C¹² Example 44pE-R-P-R-L-C-H-K-L-P-Nle-C-F-OH Disulfide C⁶-C¹² Example 45pE-R-P-R-L-C-H-K-R-P-Nle-C-F-OH Disulfide C⁶-C¹² Example 46pE-R-P-R-L-C-H-K-G-(Pipecolic acid)-Nle-C-F-OH Disulfide C⁶-C¹²Example 47 pE-R-P-R-L-C-H-K-G-P-(3-PyA)-C-F-OH Disulfide C⁶-C¹²Example 48 pE-R-P-R-L-C-H-K-G-P-Nle-C-H-OH Disulfide C⁶-C¹² Example 49pE-R-P-R-L-C-H-K-G-P-Nle-C-E-OH Disulfide C⁶-C¹² Example 50pE-R-P-R-L-C-H-K-G-P-Nle-C-OH Disulfide C⁶-C¹² Example 51pE-R-P-R-L-C-H-K-G-P-Nle-hC-F-OH Disulfide C⁶-C¹² Example 52pE-R-P-R-L-hC-H-K-G-P-Nle-hC-F-OH Disulfide C⁶-C¹² Example 53pE-R-P-R-L-c-H-K-G-P-Nle-C-F-OH Disulfide C⁶-C¹² Example 54pE-R-P-R-L-C-H-K-G-P-Nle-(D-hC)-F-OH Disulfide C⁶-C¹² Example 55pE-R-P-R-L-(D-hC)-H-K-G-P-Nle-(D-hC)-F-OH Disulfide C⁶-C¹² Example 56pE-R-P-R-L-C-H-K-G-P-Nle-c-F-OH Disulfide C⁶-C¹² Example 57pE-R-P-R-L-c-H-K-G-P-Nle-c-F-OH Disulfide C⁶-C¹² Example 58Myristoy1-O2Oc-O2Oc-Q-R-P-R-L-C-H-K-G-P-Nle-C-f- Disulfide C⁶-C¹² OHExample 59 Myristoyl-O2Oc-O2Oc-O2Oc-Q-R-P-R-L-C-H-K-G-P-Disulfide C⁶-C¹² Nle-C-f-OH Example 60Myristoy1-O2Oc-O2Oc-O2Oc-O2Oc-Q-R-P-R-L-C-H-K- Disulfide C⁶-C¹²G-P-Nle-C-f-OH Example 61 pE-R-P-R-L-C-H-K(Myristoyl)-G-P-Nle-C-F-OHDisulfide C⁶-C¹² Example 62 pE-R-P-R-L-C-H-K-G-P-Nle-C-F-NH₂Disulfide C⁶-C¹² Example 63 pE-R-P-R-L-C-H-K-G-P-Nle-C-NH₂Disulfide C⁶-C¹² Example 64 pE-R-P-R-L-C-H-K-G-P-Nle-C-F-OH—S—CH₂—C(═O)CH₂—S- [C⁶-C¹²] Example 65 pE-R-P-R-L-C-H-K-G-P-Nle-C-F-01-1Monosulfide C⁶-C¹² Example 66 pE-R-P-R-L-C-H-K-G-P-Nle-C-F-OH—S—CH₂—C(═Z)—CH₂—S- [C⁶-C¹²]

Analytical Methods 1) HPLC—Analytical Method A

-   -   Column: Bischoff UHC-640 (53×4.0 mm) with ProntoSil 120-3-C18-H,        3 μm; Part n^(o): 0604F185PS030    -   Eluent A: 0.07% TFA in water/Eluent B: 0.1% TFA in ACN    -   Flow: 1.5 ml/min    -   Temperature: 40° C.    -   Gradient:

Time [min] A [%] B [%] 0.0 90 10 9.5 0 100 12.0 0 100 12.2 90 10

2a) UPLC-MS—Analytic Method B

-   -   Waters Acquity UPLC® BEH C18, 1.7 μm, 2.1×50 mm; Part n^(o):        186002350    -   Eluent A: 0.1% FA in water; Eluent B: 0.1% FA in ACN    -   Flow: 0.7 ml/min    -   Temperature: 40° C.    -   Gradient:

Time [min] A [%] B [%] 0.0 80 20 1.0 75 25 4.2 10 90 4.3 0 100 4.6 80 20

2 b) UPLC-MS—Analytic Method C

-   -   Waters Acquity UPLC® BEH C18, 1.7 μm, 2.1×50 mm; Part n^(o):        186002350    -   Eluent A: 0.1% FA in water; Eluent B: 0.1% FA in ACN    -   Flow: 0.7 ml/min    -   Temperature: 40° C.    -   Gradient:

Time [min] A [%] B [%] 0.0 99 1 1.0 97 3 3.5 50 50 4.0 10 90 4.3 0 1004.6 80 20

The analytical data for peptides of Examples 1 to 63 are summarized inTable 5 and was generated using the analytical methods described supra.

TABLE 5 Mass spectrometry HPLC [M + 2H]²⁺ [M + 3H]³⁺ [M + 2H]²⁺ [M +3H]³⁺ Peptide t_(R) [min] Meth. (measured) (measured) Meth. (calc.)(calc.) Example 1 4.16 A 766.3 511.2 C 766.4 511.3 Example 2 4.18 A773.5 515.8 C 773.4 516.0 Example 3 4.14 A 506.6 C 759.4 506.6 Example 44.15 A 752.4 501.9 C 752.4 501.9 Example 5 3.70 A 484.5 C 726.4 484.6Example 6 3.84 A 484.5 C 726.4 484.6 Example 7 3.85 A 553.6 C 829.9553.6 Example 8 3.43 A 768.1 512.4 C 768.4 512.6 Example 9 3.77 A 495.2C 742.4 495.3 Example 3.74 A 742.5 495.1 C 742.4 495.3 10 Example 3.61 A742.9 495.2 C 742.4 495.3 11 Example 3.62 A 497.8 C 746.4 497.9 12Example 3.49 A 768.3 512.5 C 768.4 512.6 13 Example 4.14 A 808.5 539.2 C808.4 539.3 14 Example 3.99 A 773.4 515.8 C 773.4 515.9 15 Example 3.36A 475.5 C 712.9 475.6 16 Example 3.28 A 527.5 C 790.9 527.6 17 Example3.36 A 512.5 C 768.4 512.6 18 Example 4.38 A 756.0 504.2 C 755.9 504.319 Example 3.17 A 782.6 522.0 C 782.4 521.9 20 Example 3.45 A 512.0 C767.4 511.9 21 Example 6.12 A 585.6 B 878.0 585.7 22 Example 6.46 A883.2 588.9 B 883.0 589.0 23 Example 5.20 A 646.0 B 968.6 646.0 24Example 5.18 A 627.3 B 940.5 627.3 25 Example 5.11 A 567.0 B 850.0 567.026 Example 5.44 A 570.0 B 855.0 570.3 27 Example 4.16 A 723.7 C 723.8482.9 28 Example 3.85 A 753.0 502.5 C 753.3 502.6 29 Example 3.39 A512.5 C 768.4 512.6 30 Example 4.08 A 763.8 509.4 C 763.9 509.6 31Example 3.59 A 754.8 503.6 C 754.9 503.6 32 Example 3.36 A 512.5 C 768.4512.6 33 Example 3.14 A 522.8 C 783.9 522.9 34 Example 3.36 A 518.5 C777.4 518.6 35 Example 3.91 A 763.8 509.4 C 763.9 509.6 36 Example 3.05A 517.5 C 775.9 517.6 37 Example 3.67 A 768.7 512.8 C 768.9 512.9 38Example 3.47 A 531.7 C 797.4 531.9 39 Example 3.60 A 754.9 503.6 C 754.9503.6 40 Example 3.91 A 549.1 C 823.4 549.3 41 Example 3.10 A 769.2513.1 C 769.4 513.2 42 Example 3.58 A 764.2 509.7 C 764.4 509.9 43Example 3.82 A 531.1 C 796.4 531.3 44 Example 3.16 A 545.5 C 817.9 545.645 Example 3.54 A 517.1 C 775.4 517.3 46 Example 2.53 A 524.1 C 785.9524.3 47 Example 2.49 A 509.2 C 763.4 509.3 48 Example 2.73 A 759.3506.5 C 759.4 506.6 49 Example 2.72 A 694.5 C 694.8 463.6 50 Example3.38 A 517.1 C 775.4 517.3 51 Example 3.45 A 521.9 C 782.4 521.9 52Example 3.52 A 768.4 512.5 C 768.4 512.6 53 Example 3.43 A 775.3 517.1 C775.4 517.3 54 Example 3.83 A 782.3 521.8 C 782.4 521.9 55 Example 3.42A 768.1 512.4 C 768.4 512.6 56 Example 3.66 A 768.3 512.4 C 768.4 512.657 Example 5.68 A 685.0 B 1027.1 685.0 58 Example 5.58 A 733.4 B 1099.6733.4 59 Example 5.55 A 781.8 B 1172.1 781.8 60 Example 3.18 D 874.5582.9 D 873.5 585.7 61 Example 3.22 A 512.3 C 767.9 512.3 62 Example2.71 A 694.3 463.1 C 694.4 463.2 63

General Synthesis Procedures

1) Loading of First Amino Acid onto 2-Chlorotrityl Chloride Resin andFmoc-Removal

2-Chlorotrityl chloride resin (1 eq., 1.0-1.6 mmol/g) was washedthoroughly with DCM. The desired amino acid (typically 0.5-2 eq.relative to the resin, considering 1.6 mmol/g loading) was dissolved inDCM (approx. 10 mL per gram of resin) and DIPEA (4 eq. relative to theresin, considering 1.6 mmol/g loading). The solution was added to theresin and the suspension was shaken at it for 19 h. The resin wasdrained and then thoroughly washed sequentially with DCM/MeOH/DIPEA(17:2:1), DCM, DMA, DCM.

For Fmoc removal and determination of the loading the resin was shakenrepeatedly with piperidine/DMA (1:4) or 4-methylpiperidine/DMA (1:4)(12×10 mL per gram of initial resin) and washed with DMA (2×10 mL pergram of initial resin). The combined solutions were diluted with MeOH toa volume V of 250 mL per gram of initial resin. A 2 mL aliquot (V_(a))of this solution was diluted further to 250 mL (V_(t)) with MeOH. The UVabsorption was measured at 299.8 nm against a reference of MeOH, givingabsorption A. The resin was thoroughly washed sequentially with DMA,DCM, DMA, DCM and dried in high vacuum at 40° C., affording m g ofresin.

The loading of the resin is calculated according to the formula:

Loading [mol/g]=(A×V _(t) ×V)/(d×ε×V _(a) ×m)

(with d: width of cuvette; ε=7800 L mol⁻¹ cm⁻¹)

2) Solid Phase Peptide Synthesis on Prelude™ Synthesizer 2a) SynthesisCycle A

The resin was washed with DMA. Fmoc was removed by repetitive treatmentwith 4-methylpiperidine/DMA (1:4). The resin was washed with DMA.Coupling was done by addition of the Fmoc-amino acid (3 eq.; 0.2 Msolution in NMP), HCTU (3 eq.; 0.3 M solution in NMP), and DIPEA (3.3eq.; 0.66 M solution in NMP) followed by mixing of the suspension withnitrogen at it for typically 15 min to 4 h depending on the specificrequirements. After washing with DMA the coupling step was typicallyrepeated 1 to 3 times depending on the specific requirements. Afterwashing with DMA capping was performed by addition of a mixture ofAc₂O/pyridine/DMA (1:1:8) and subsequent mixing of the suspension at rt.The resin was washed with DMA.

2b) Synthesis Cycle B

The resin was washed with DMA. Fmoc was removed by repetitive treatmentwith piperidine/DMA (1:4). The resin was washed with DMA. Coupling wasdone by addition of the Fmoc-amino acid (3 eq.; 0.3 M solution in NMP),HCTU (3 eq.; 0.3 M solution in NMP), and DIPEA (4.5 eq.; 0.9 M solutionin NMP) followed by mixing of the suspension with nitrogen at rt fortypically 15 min to 4 h depending on the specific requirements. Afterwashing with DMA the coupling step was typically repeated 1 to 3 timesdepending on the specific requirements. After washing with DMA cappingwas performed by addition of a mixture of Ac₂O/pyridine/DMA (1:1:8) andsubsequent mixing of the suspension at rt. The resin was washed withDMA.

2c) Synthesis Cycle C

The resin was washed with DMA. Fmoc was removed by repetitive treatmentwith piperidine/DMA (1:4). The resin was washed with DMA. Coupling wasdone by addition of the Fmoc-amino acid (3 eq.; 0.3 M solution in NMP),HCTU (3 eq.; 0.3 M solution in NMP), and DIPEA (6 eq.; 0.9 M solution inNMP) followed by mixing of the suspension with nitrogen at it fortypically 15 min to 4 h depending on the specific requirements. Afterwashing with DMA the coupling step was typically repeated 1 to 3 timesdepending on the specific requirements. After washing with DMA cappingwas performed by addition of a mixture of Ac₂O/pyridine/DMA (1:1:8) andsubsequent mixing of the suspension at rt. The resin was washed withDMA.

2d) Synthesis Cycle D

The resin was washed with DMA. Fmoc was removed by repetitive treatmentwith 4-methylpiperidine/DMA (1:4). The resin was washed with DMA.Coupling was done by addition of a mixture of the Fmoc-amino acid andOxyma Pure (3 eq. each; 0.2 M of both in NMP) and DIC (3eq.; 0.3 Msolution in NMP) followed by mixing of the suspension with nitrogen atit for typically 15 min to 4 h depending on the specific requirements.After washing with DMA the coupling step was typically repeated 1 to 3times depending on the specific requirements. After washing with DMAcapping was performed by addition of a mixture of Ac₂O/pyridine/DMA(1:1:8) and subsequent mixing of the suspension at rt. The resin waswashed with DMA.

3) Cleavage from Resin with or without Concomitant Removal of ProtectingGroups

3a) Cleavage Method A

The resin (0.1 mmol) was shaken at it for 2 h with 95% aq. TFA/EDT/TIS(95:2.5:2.5) (3 mL). The cleavage solution was filtered off, and freshsolution was added (3 mL). The suspension was shaken at it for 1 h thenthe cleavage solution was filtered off. Fresh solution was added (3 mL)and the suspension was shaken at it for 1 h. The cleavage solution wasfiltered off. The combined cleavage solutions were poured slowly onto amixture of cold heptane/diethyl ether (1:1) (35 mL), giving aprecipitate. The suspension was centrifuged and the supernatant pouredoff. The residue was washed with cold heptane/diethyl ether (1:1) (10mL), the suspension was centrifuged and the supernatant was poured off.The solid was dried in high vacuum.

3b) Cleavage Method B

The resin (0.1 mmol) was treated with 95% aq. TFA/EDT (4:1) (0.75 mL)and the suspension was shaken at it for 1 h. A mixture of 95% aq. TFA(2.18 mL) and TIS (75 μL) was added and shaking at it was resumed for 1h. The cleavage solution was filtered off then 95% aq. TFA/EDT/TIS(95:2.5:2.5) (3 mL) was added to the resin and the suspension was shakenat rt for 1 h. The cleavage solution was filtered off and collected andfresh solution was added (3 mL). The suspension was shaken at rt for 1 hthen the cleavage solution was filtered off. The combined cleavagesolutions were poured onto cold heptane/diethyl ether (1:1) (35 mL). Theprecipitate thus formed was left to settle, centrifuged then thesupernatant was carefully poured off. The precipitate was washed oncewith cold heptane/diethyl ether (1:1) (10 mL), the suspension wascentrifuged and the supernatant was poured off. The residue was dried inhigh vacuum.

3c) Cleavage Method C

HFIP/DCM (30:70) (5 mL) was added to the resin (0.1 mmol) and thesuspension was stirred at rt for 1.5 h. The cleavage solution wasfiltered off and collected and fresh HFIP/DCM (30:70) (5 mL) was added.The suspension was stirred at rt for 30 min. The cleavage solution wasfiltered off and collected. The resin was washed with DCM (2×5 mL) whichwas also collected. The combined cleavage and washing solutions wereconcentrated to dryness in high vacuum. The residue was lyophilized fromtBuOH/H₂O (1:1).

4) Cyclization Methods 4a) Cyclization Method A (Disulfide Formation)

The fully deprotected linear precursor peptide was dissolved in H₂O/DMSO(9:1) or (4:1) to give typically a concentration of 1-15 mg/mL. Thereaction mixture was then stirred at rt for typically 40 h depending onthe requirements and then concentrated to dryness in high vacuum.

4b) Cyclization Method B (Disulfide Formation)

The fully deprotected linear precursor peptide (1 eq.) was dissolved inH₂O to give typically a concentration of 10 mg/mL. A solution of 50 mMI₂ in AcOH (1.2 eq.) was added in one portion to the stirred solutionand the reaction was stirred for 10 min at rt. 0.5 M Ascorbic acid inH₂O (1.5 eq) was added to quench the excess of 12. The solution wasconcentrated to near dryness in vacuo.

4c) Cyclization Method C (Selective Formation of Two Disulfides)

The partially protected linear precursor peptide (1 eq.) (two cysteineswere protected with Acm and two cysteines unprotected) was dissolved inAcOH/H₂O (4:1) to give typically a concentration of 1 mg/mL. 50 mM I₂ inAcOH (2 eq.) was added and the reaction mixture was stirred at rt for 1h. Further 50 mM I₂ in AcOH (10 eq.) was added portionwise over 4 h.After 21 h, the reaction mixture was concentrated to near dryness invacuo and 1 M ascorbic acid in H₂O was added in excess to quenchunreacted I₂.

4d) Cyclization Method D (Lactam Formation Between Side Chains)

The fully deprotected linear precursor peptide (1 eq.) and HATU (1.5eq.) were dissolved in NMP (peptide concentration: typically) mmol/L).DIPEA (3 eq.) was added and the solution stirred at it for 90 min. Thereaction mixture was concentrated to dryness in vacuo.

4e) Cyclization Method E (Lactam Formation Between Side Chain andC-Terminus)

A solution of the peptide (1 eq.), HATU (1.3 eq.) and HOAt (1.3 eq.) inDMF (peptide concentration: 2.6 mmol/L) was treated with 2,6-lutidine(20 eq.) and the reaction was stirred at rt for 2 h. The reactionmixture was concentrated to dryness in vacuo.

In the following the syntheses of representative examples are described.

Example 1 Synthesis of pE-R-P-R-L-K-H-F-G-P-Nle-D-Phenethylamine (LactamK⁶-D¹²)

Preparation of Intermediate 1a

(Assembly of Linear Peptide)

Phenethylamine-BAL-PS resin (167 mg, 0.100 mmol) was subjected to solidphase peptide synthesis on the Prelude™ peptide synthesizer. Couplingwas performed as follows:

Number of couplings × Synthesis Coupling AA Reaction time cycle 1 D(tBu)2 × 4 h C 2 Nle 1 × 3 h C 3 P 2 × 45 min C 4 G 2 × 90 min C 5 F 1 × 3 hC 6 H(Trt) 2 × 45 min C 7 K(Boc) 2 × 4 h C 8 L 4 × 1 h C 9 R(Pbf) 4 × 1h C 10 P 2 × 90 min C 11 R(Pbf) 4 × 1 h C 12 pE 2 × 90 min C

Preparation of Intermediate 1b

(Cleavage from the Resin with Concomitant Protecting Group Removal thenPurification)

A mixture of 95% aq. TFA/EDT/TIS (95:2.5:2.5) (2 mL) was added toIntermediate 1a (0.1 mmol) and the suspension was shaken at rt for 2.5h. The cleavage solution was filtered off, and fresh cleavage solution(2 mL) was added. The suspension was shaken at rt for 45 min then thecleavage solution was filtered off. Fresh solution (2 mL) was added andthe suspension was shaken at it for 45 min. The cleavage solution wasfiltered off and the resin was washed with 95% aq. TFA (1 mL). Thecombined cleavage solutions were poured onto a mixture of coldheptane/diethyl ether (1:1) (35 mL), giving a precipitate. Thesuspension was centrifuged and the supernatant poured off. The residuewas washed with cold heptane/diethyl ether (1:1) (20 mL), the suspensionwas centrifuged and the supernatant was poured off. The solid was driedin high vacuum. The crude was purified by preparative HPLC andlyophilized from ACN/H₂O to afford Intermediate 1b as a white solid intwo batches of different qualities: Batch A (35.9 mg (98% purity), 0.018mmol) and batch B (52.9 mg (80% purity), 0.021 mmol).

Preparation of Example 1

(Cyclization and Purification)

Both batches from the previous step were treated separately followingthe same protocol:

Batch A: A solution of the peptide (35.9 mg (98% purity), 0.018 mmol)and HATU (10.0 mg, 0.026 mmol) in NMP (18 mL) and DIPEA (9.2 μL, 0.053mmol) was stirred at it for 2 h.

Batch B: A solution of the peptide (52.9 mg (80% purity), 0.021 mmol)and HATU (14.5 mg, 0.038 mmol) in NMP (26 mL) and DIPEA (13.0 μL, 0.076mmol) was stirred at II for 2 h.

Each of the batches was concentrated to dryness in vacuo. The productwas isolated by preparative HPLC. Pure fractions of both purificationswere combined and lyophilized from ACN/H₂O to give Example 1 as a whitesolid (52.0 mg, 0.025 mmol).

The pure product was analyzed by analytical HPLC (Analytical method A:t_(R)=4.16 min) and UPLC-MS (Analytical method C; measured:[M+3]³⁺=511.2; calculated: [M+3]³⁺=511.3).

Example 5 Synthesis pE-R-P-R-L-K-F-K-G-P-Nle-F (Lactam K⁶-C-Terminus)

Preparation of Intermediate 5a

(Loading of 2-Chlorotrityl Chloride Resin with Fmoc-F-OH, Fmoc Removaland Determination of the Loading of the Resin)

2-Chlorotrityl chloride resin (10.0 g, 16.0 mmol) was reacted with asolution of Fmoc-F-OH (6.24 g, 32.0 mmol) in DCM (100 mL) and DIPEA(11.2 mL, 64.0 mmol) in analogy to the general procedure described aboveto give Intermediate 5a (12.8 g, loading=0.79 mmol/g).

Preparation of Intermediate 5b

(Assembly of Linear Peptide)

Intermediate 5a (0.100 mmol) was subjected to solid phase peptidesynthesis on the Prelude™ peptide synthesizer. Coupling was performed asfollows:

Number of couplings × Synthesis Coupling AA Reaction time cycle 2 Nle 2× 90 min B 3 P 2 × 30 min B 4 G 2 × 90 min B 5 K(ivDde) 2 × 30 min B 6 F2 × 30 min B 7 K(Boc) 4 × 1 h B 8 L 2 × 30 min B 9 R(Pbf) 4 × 1 h B 10 P2 × 90 min B 11 R(Pbf) 4 × 1 h B 12 pE 2 × 90 min B

Preparation of Intermediate 5c

(Removal of ivDde and Cleavage from the Resin)

Intermediate 5b (0.100 mmol) was treated six times for 10 min with asolution of hydrazine monohydrate (0.081 mL, 1.67 mmol) in DMA (4 mL).Then the resin was treated three times for 20 min with a solution ofhydrazine monohydrate (0.081 mL, 1.67 mmol) in THF (4 mL). The resin waswashed with DCM (3×). HFIP/DCM (30:70) (5 mL) was added to the resin(0.100 mmol) and the suspension was stirred at it for 1.5 h. Thecleavage solution was filtered off and fresh HFIP/DCM (30:70) (5 mL) wasadded. The suspension was stirred at it for 30 min. The cleavagesolution was filtered off. The resin was washed with DCM (2×5 mL). Thecombined cleavage and washing solutions were concentrated to dryness invacuo. The residue was lyophilized from tBuOH/H₂O (1:1) to giveIntermediate 5c (187 mg, 0.090 mmol).

Preparation of Example 5

(Cyclization and Removal of Protecting Groups)

A solution of Intermediate 5c (187 mg, 0.090 mmol), HATU (44.6 mg, 0.117mmol) and HOAt (16.0 mg, 0.117 mmol) in DMF (35 mL) was treated with2,6-lutidine (0.210 mL, 1.80 mmol) and the reaction was stirred at rtfor 2 h. The reaction mixture was concentrated to dryness in vacuo. Theresidue was dissolved in 95% aq. TFA/EDT/TIS (95:2.5:2.5) (5 mL) and thesolution was stirred at rt for 2.5 h. The cleavage solution was pouredonto cold heptane/diethyl ether (1:1) (30 mL), giving a precipitate. Thesuspension was centrifuged and the supernatant poured off. The residuewas washed with cold heptane/diethyl ether (1:1) (10 mL), the suspensionwas centrifuged and the supernatant was poured off. The washing step wasrepeated once. The residue was dried in high vacuum. The product wasisolated by preparative HPLC and lyophilized from ACN/H₂O to affordExample 5 as a white solid (41.4 mg, 0.023 mmol).

The pure product was analyzed by analytical HPLC (Analytical method A:t_(R)=3.70 min) and UPLC-MS (Analytical method C; measured:[M+3]³⁺=484.5; calculated: [M+3]³⁺=484.6).

Example 7 Synthesis Q-R-P-R-L-C-F-K-G-P-Nle-C-F-G-G (LactamN-Terminus-C-Terminus)

Preparation of Intermediate 7a

(Loading of 2-Chlorotrityl Chloride Resin with Fmoc-Gly-OH, Fmoc Removaland Determination of the Loading of the Resin)

2-Chlorotrityl chloride resin (2.00 g, 3.20 mmol) was reacted with asolution of Fmoc-Gly-OH (0.476 g, 1.60 mmol) in DCM (20 mL) and DIPEA(2.24 mL, 12.8 mmol) in analogy to the general procedure described aboveto give Intermediate 7a (2.22 g; loading=0.68 mmol/g).

Preparation of Intermediate 7b

(Assembly of Linear Peptide and Fmoc Removal)

Intermediate 7a (147 mg, 0.100 mmol) was subjected to solid phasepeptide synthesis on the Prelude™ peptide synthesizer. Coupling wasperformed as follows:

Number of couplings × Synthesis Coupling AA Reaction time cycle 1 G 2 ×30 min B 2 F 2 × 30 min B 3 C(Trt) 2 × 30 min B 4 Nle 2 × 90 min B 5 P 2× 30 min B 6 G 2 × 90 min B 7 K(Boc) 2 × 30 min B 8 F 2 × 30 min B 9C(Trt) 2 × 30 min B 10 L 2 × 30 min B 11 R(Pbf) 4 × 1 h B 12 P 2 × 90min B 13 R(Pbf) 4 × 1 h B 14 Q(Trt) 2 × 90 min B

After assembly of the peptide Fmoc was removed by repetitive treatmentwith piperidine/DMA (1:4). The resin was washed with DMA to affordIntermediate 7b (0.100 mmol).

Preparation of Intermediate 7c

(HFIP Cleavage from the Resin)

HFIP/DCM (30:70) (3 mL) was added to Intermediate 7b (0.100 mmol) andthe suspension was shaken at rt for 1.5 h. The cleavage solution wasfiltered off and fresh HFIP/DCM (30:70) (3 mL) was added. The suspensionwas shaken at rt for 30 min. The cleavage solution was filtered off. Theresin was washed with DCM (2×3 mL). The combined cleavage and washingsolutions were concentrated to dryness in vacuo. The residue waslyophilized from tBuOH/H₂O (1:1) to give Intermediate 7c (203 mg, 0.067mmol).

Preparation of Intermediate 7d

(Backbone Cyclization)

A solution of Intermediate 7c (203 mg, 0.067 mmol), HATU (33.3 mg, 0.088mmol) and HOAt (11.9 mg, 0.088 mmol) in DMF (40 mL) was treated with2,6-lutidine (0.157 ml, 1.35 mmol) and the reaction was stirred at itfor 2 h. The reaction mixture was concentrated to dryness in vacuo toafford Intermediate 7d (0.067 mmol).

Preparation of Intermediate 7e

(Removal of Protecting Groups then Purification)

A mixture of 95% aq. TFA/EDT/TIS (95:2.5:2.5) (3 mL) was added toIntermediate 7d (0.067 mmol) and the suspension was shaken at it for 2.5h. The solution was poured onto a mixture of cold heptane/diethyl ether(1:1) (30 mL), giving a precipitate. The suspension was centrifuged andthe supernatant poured off. The residue was washed with coldheptane/diethyl ether (1:1) (10 mL), the suspension was centrifuged andthe supernatant was poured off. The washing step was repeated once. Thesolid was dried in high vacuum. The crude was purified by preparativeHPLC and lyophilized from ACN/H₂O to afford Intermediate 7e as a whitesolid (33.6 mg, 0.017 mmol).

Preparation of Example 7

(Cyclization and Purification)

Intermediate 7e (33.6 mg, 0.017 mmol) was dissolved in H₂O/DMSO (9:1)(30 mL). The reaction mixture was stirred at rt for 40 h thenconcentrated to dryness in vacuo. The crude was purified by preparativeHPLC and lyophilized from ACN/H₂O to afford Example 7 as a white solid(21.0 mg; 0.010 mmol).

The pure product was analyzed by analytical HPLC (Analytical method A:t_(R)=3.85 min) and UPLC-MS (Analytical method C; measured:[M+3]³⁺=553.6; calculated: [M+3]³⁺=553.6).

Example 8 Synthesis of pE-R-P-R-L-C-H-K-G-P-Nle-C-F-OH (DisulfideC⁶-C¹²)

Preparation of Intermediate 8a

(Loading of 2-Chlorotrityl Chloride Resin with Fmoc-F-OH, Fmoc Removaland Determination of the Loading of the Resin)

2-Chlorotrityl chloride resin (40.0 g, 64.0 mmol) was washed with DCM(3×). A solution of Fmoc-F-OH (24.8 g, 64.0 mmol) in DCM (400 mL) andDIPEA (44.7 mL, 256 mmol) was added and the suspension was shaken for 22h at rt. The resin was washed thoroughly with DCM/MeOH/DIPEA (17:2:1)(3×), DCM (3×), DMA (3×), DCM (3×).

The resin was then treated four times for 10 min with a mixture ofpiperidine/DMA (1:4) (400 mL) followed by washing with DMA (2×180 ml).The piperidine/DMA solutions and DMA washing solutions were collectedfor determination of the loading of the resin. 1 mL of the combinedsolutions was diluted to 500 mL with MeOH and the UV absorption at 299.8nm was measured to be A=0.368. This corresponds to an Fmoc amount of46.2 mmol.

The resin was washed thoroughly with DCM (3×), DMA (3×), DCM (3×) anddried in vacuo to give Intermediate 8a (50.7 g; loading=0.91 mmol/g).

Preparation of Intermediate 8b

(Assembly of Linear Peptide)

Intermediate 8a (2.64 g, 2.40 mmol) was subjected to solid phase peptidesynthesis on the Prelude™ peptide synthesizer. Coupling was performed asfollows:

Number of couplings × Synthesis Coupling AA Reaction time cycle 1 C(Trt)2 × 30 min D 2 Nle 2 × 15 min A 3 P 2 × 15 min A 4 G 2 × 30 min A 5K(Boc) 2 × 15 min A 6 H(Trt) 2 × 15 min A 7 C(Trt) 2 × 60 min D 8 L 2 ×15 min A 9 R(Pbf) 4 × 1 h A 10 P 2 × 15 min A 11 R(Pbf) 4 × 1 h A 12 pE2 × 15 min A

Preparation of Intermediate 8c

(Cleavage from the Resin with Concomitant Protecting Group Removal)

Intermediate 8b (2.40 mmol) was carefully washed with DCM (4×). Amixture of 95% aq. TFA/EDT/TIS (95:2.5:2.5) (50 mL) was added and thesuspension was shaken at rt for 1 h. The cleavage solution was filteredoff, and fresh cleavage solution (35 mL) was added. The suspension wasshaken at rt for 1 h then the cleavage solution was filtered off. Freshsolution (35 mL) was added and the suspension was shaken at rt for 1 h.The cleavage solution was filtered off. The combined cleavage solutionswere poured slowly onto a stirred mixture of cold heptane/diethyl ether(1:1) (500 mL), giving a precipitate. The suspension was stirred at rtfor 2 h and then the precipitate was allowed to settle down. Thesupernatant was sucked off with a frit. The residue was washed with coldheptane/diethyl ether (1:1) (2×100 mL), the supernatant was sucked offwith a frit. The solid was dried in high vacuum to afford Intermediate8c as an off-white solid (3.75 g, 1.88 mmol).

Preparation of Example 8

(Cyclization and Purification)

Intermediate 8c (3.75 g, 1.88 mmol) was dissolved in H₂O (375 mL). Asolution of 50 mM 12 in AcOH (45.1 mL, 2.26 mmol) was added in oneportion to the stirred solution and the solution was stirred for 10 minat rt. 0.5 M Ascorbic acid in H₂O (5.64 mL, 2.82 mmol) was added toquench the excess of I₂. The solution was concentrated to near dryness.The reaction was performed in two portions: 0.188 mmol scale and 1.69mmol scale. The crudes were combined for purification. The crude waspurified by preparative HPLC and lyophilized from ACN/H₂O to affordExample 8 as a white solid (1.53 g, 0.767 mmol).

The pure product was analyzed by analytical HPLC (Analytical method C:t_(R)=3.43 min) and UPLC-MS (Analytical method B; measured:[M+3]³⁺=512.4; calculated: [M+3]³⁺=512.6).

Alternatively, The crude polypeptide of Example 8 was dissolved in water(500 mL of water/mmol of polypeptide) and was converted into the acetatesalt with the aid of an ion exchange resin (i.e. Amberlite IRA-67(Acetate-Form)(200 g/mmol of polypeptide) and purified by preparativeHPLC (C8 modified reversed phase silica gel from Daisogel, gradient:ACN/H₂O: 3% ACN and 97% [mixture 0.3% Acetic acid/water] up to 12% ACNand 88% [mixture 0.3% Acetic acid/water]) and lyophilized to afford anacetate salt of Example 8 as a white solid (60-100% yield).

The salt stoichiometry was evaluated based on the analysis of the aceticacid content (ion chromatography) and water content and was determinedto range between 1:3 and 1:4 (polypeptide: acetate).

Example 26 Synthesis of pE-R-P-R-L-C-H-K-G-P-Nle-C-K(Lauroyl)-OH(Disulfide C⁶-C¹²)

Preparation of Intermediate 26a

(Loading of 2-Chlorotrityl Chloride Resin with Fmoc-K(ivDde)-OH, FmocRemoval and Determination of the Loading of the Resin)

2-Chlorotrityl chloride resin (1.00 g, 1.60 mmol) was reacted with asolution of Fmoc-K(ivDde)-OH (1.84 g, 3.20 mmol) in DCM (10 mL) andDIPEA (1.12 mL, 6.40 mmol) in analogy to the general procedure describedabove to give Intermediate 26a (1.39 g; loading=0.75 mmol/g).

Preparation of Intermediate 26b

(Assembly of Linear Peptide)

Intermediate 26a (134 mg, 0.100 mmol) was subjected to solid phasepeptide synthesis on the Prelude™ peptide synthesizer. Coupling wasperformed as follows:

Number of couplings × Synthesis Coupling AA Reaction time cycle 1 C(Trt)2 × 30 min B 2 Nle 2 × 30 min B 3 P 2 × 30 min B 4 G 2 × 90 min B 5K(Boc) 2 × 30 min B 6 H(Trt) 2 × 30 min B 7 C(Trt) 2 × 30 min B 8 L 2 ×30 min B 9 R(Pbf) 4 × 1 h B 10 P 2 × 90 min B 11 R(Pbf) 4 × 1 h B 12 pE2 × 90 min B

Preparation of Intermediate 26c

(Removal of ivDde and Coupling of La Uric Acid)

Intermediate 26b (0.100 mmol) was washed with THF (3×). A solution ofhydrazine monohydrate (0.245 ml, 5.00 mmol) in THF (12 mL) was added andthe suspension was shaken at it for 1 h. The solution was filtered off.This step was done twice more. The resin was washed with DMA (3×), DCM(3×), DMA (2×), DCM (5×) and DMA (3×). Lauric acid (100 mg, 0.500 mmol)and HCTU (207 mg, 0.500 mmol) were dissolved in NMP (3 mL) and DIPEA(0.087 ml, 0.500 mmol). After 5 min activation the solution was added tothe resin and the suspension was shaken at it for 16 h. The reactionmixture was filtered off and the resin washed with DMA (3×), DCM (3×),DMA (3×), DCM (5×) to afford Intermediate 26c.

Preparation of Intermediate 26d

(Cleavage from the Resin with Concomitant Protecting Group Removal thenPurification)

A mixture of 95% aq. TFA/EDT/TIS (95:2.5:2.5) (3 mL) was added toIntermediate 26c (0.100 mmol) and the suspension was shaken at it for 2h. The cleavage solution was filtered off, and fresh cleavage solution(3 mL) was added. The suspension was shaken at rt for 1 h then thecleavage solution was filtered off. Fresh solution (3 mL) was added andthe suspension was shaken at it for 1 h. The cleavage solution wasfiltered off. The combined cleavage solutions were poured onto a mixtureof cold heptane/diethyl ether (1:1) (35 mL), giving a precipitate. Thesuspension was centrifuged and the supernatant poured off. The residuewas washed with cold heptane/diethyl ether (1:1) (10 mL), the suspensionwas centrifuged and the supernatant was poured off. The solid was driedin high vacuum.

The crude was purified by preparative HPLC and lyophilized from ACN/H₂Oto afford Intermediate 26d as a white solid (74.1 mg, 0.034 mmol).

Preparation of Example 26

(Cyclization and Purification)

Intermediate 2d (74.1 mg, 0.034 mmol) was dissolved in H₂O/DMSO (9:1)(74 mL). The reaction mixture was stirred at rt for 40 h thenconcentrated to dryness in vacuo. The crude was purified by preparativeHPLC and lyophilized from ACN/H₂O to afford Example 26 as a white solid(60.0 mg; 0.028 mmol).

The pure product was analyzed by analytical HPLC (Analytical method A:t_(R)=5.11 min) and UPLC-MS (Analytical method B; measured:[M+3]³⁺=567.0; calculated: [M+3]³⁺=567.0).

Example 28 Synthesis pE-R-P-C-L-C-C-K-G-P-Nle-C-F-OH (Disulfides C⁴-C⁷and C⁶-C¹²)

Preparation of Intermediate 28a

(Loading of 2-Chlorotrityl Chloride Resin with Fmoc-F-OH, Fmoc Removaland Determination of the Loading of the Resin)

2-Chlorotrityl chloride resin (10.0 g, 16.0 mmol) was reacted with asolution of Fmoc-F-OH (6.20 g, 16.0 mmol) in DCM (100 mL) and DIPEA(11.2 mL, 64.0 mmol) in analogy to the general procedure described aboveto give Intermediate 28a (11.6 g, loading=0.87 mmol/g).

Preparation of Intermediate 28b

(Assembly of Linear Peptide)

Intermediate 28a (115 mg, 0.100 mmol) was subjected to solid phasepeptide synthesis on the Prelude™ peptide synthesizer. Coupling wasperformed as follows:

Number of couplings × Synthesis Coupling AA Reaction time cycle 1 C(Trt)2 × 15 min B 2 Nle 2 × 15 min B 3 P 2 × 15 min B 4 G 2 × 90 min B 5K(Boc) 2 × 15 min B 6 C(Acm) 2 × 15 min B 7 C(Trt) 2 × 15 min B 8 L 2 ×15 min B 9 C(Acm) 2 × 15 min B 10 P 2 × 15 min B 11 R(Pbf) 4 × 1 h B 12pE 2 × 15 min B

Preparation of Intermediate 28c

(Cleavage from the Resin with Concomitant Partial Protecting GroupRemoval)

Intermediate 28b (0.100 mmol)) was carefully washed with DCM (4×). Amixture of 95% aq. TFA/EDT (4:1) (0.750 mL) was added and the suspensionwas shaken at it for 1 h. A mixture of TFA/H₂O (95:5) (2.18 mL) and TIS(75 μL) was added to the suspension and shaking at it was continued for1 h. The cleavage solution was filtered off and a mixture of 95% aq.TFA/EDT/TIS (95:2.5:2.5) (3 mL) was added to the resin. The suspensionwas shaken at it for 1 h the cleavage solution was filtered off. Freshsolution was added (3 mL) and the suspension was shaken at it for 1 h.The cleavage solution was filtered off. The combined cleavage solutionswere poured onto cold heptane/diethyl ether (1:1) (35 mL), giving aprecipitate. The suspension was centrifuged and the supernatant pouredoff. The residue was washed with cold heptane/diethyl ether (1:1) (10mL), the suspension was centrifuged and the supernatant was poured off.The washing step was repeated once. The residue was dried in highvacuum. The crude product was purified by preparative HPLC andlyophilized from ACN/H₂O to afford Intermediate 28c as a white solid(51.1 mg, 0.028 mmol).

Preparation of Example 28

(One-Pot Formation of Two Disulfides)

Intermediate 28c (51.1 mg, 0.028 mmol) was dissolved in AcOH (48 mL) andH₂O (12 mL). A 50 mM solution of I₂ in AcOH (1.12 mL, 56 μmol) was addedand the yellow solution was stirred at it. Further 50 mM I₂ in AcOH(5.61 mL, 0.281 mmol) was added portion wise over 4 h. After 21 h, thereaction mixture was concentrated to 2 mL in vacuo and 1 M ascorbic acidin H₂O (6 mL) was added to quench the excess of I₂. The product wasisolated by preparative HPLC and lyophilized from ACN/H₂O to affordExample 28 as a white solid (19.3 mg, 0.012 mmol).

The pure product was analyzed by analytical HPLC (Analytical method A:t_(R)=4.16 min and UPLC-MS (Analytical method C; measured:[M+2]²⁺=723.7; calculated: [M+2]²⁺=723.8).

Example 62 Synthesis of pE-R-P-R-L-C-H-K-G-P-Nle-C-F-NH2 (DisulfideC⁶-C¹²)

Preparation of Intermediate 62a

(Assembly of Linear Peptide)

Fmoc protected Rink-Amide-AM-PS-resin (217 mg, 0.100 mmol) was subjectedto solid phase peptide synthesis on the Prelude™ peptide synthesizer.Coupling was performed as follows:

Number of couplings × Synthesis Coupling AA Reaction time cycle 1 F 2 ×15 min A 2 C(Trt) 2 × 30 min D 3 Nle 2 × 15 min A 4 P 2 × 15 min A 5 G 2× 30 min A 6 K(Boc) 2 × 15 min A 7 H(Trt) 2 × 15 min A 8 C(Trt) 2 × 1 hD 9 L 2 × 15 min A 10 R(Pbf) 4 × 1 h A 11 P 2 × 15 min A 12 R(Pbf) 4 × 1h A 13 pE 2 × 15 min A

Preparation of Intermediate 62b

(Cleavage from the Resin with Concomitant Protecting Group Removal)

A mixture of 95% aq. TFA/EDT/TIS (95:2.5:2.5) (3 mL) was added toIntermediate 62a (0.1 mmol) and the suspension was shaken at rt for 1.5h. The cleavage solution was filtered off, and fresh cleavage solution(2 mL) was added. The suspension was shaken at rt for 45 min then thecleavage solution was filtered off. Fresh solution (2 mL) was added andthe suspension was shaken at it for 45 min. The combined cleavagesolutions were poured onto a mixture of cold heptane/diethyl ether (1:1)(35 mL), giving a precipitate. The suspension was centrifuged and thesupernatant poured off. The residue was washed with cold heptane/diethylether (1:1) (10 mL), the suspension was centrifuged and the supernatantwas poured off. The solid was dried in high vacuum. The crude productIntermediate 62b was used in the next step without purification.

Preparation of Example 62

(Cyclization and Purification)

Intermediate 62b (0.100 mmol) was dissolved in H₂O (20 mL). A solutionof 50 mM I₂ in AcOH (2.4 mL, 0.120 mmol) was added in one portion to thestirred solution and the solution was stirred for 30 min at rt. 0.5 MAscorbic acid in H₂O (0.30 mL, 0.300 mmol) was added to quench theexcess of I₂. The solution was concentrated to near dryness. The crudewas purified by preparative HPLC and lyophilized from ACN/H₂O to affordExample 62 as a white solid (50.5 mg, 0.025 mmol).

The pure product was analyzed by analytical HPLC (Analytical method C:t_(R)=3.22 min) and UPLC-MS (Analytical method C; measured:[M+3]³⁺=512.3; calculated: [M+3]³⁺=512.3).

The other examples were synthesized in analogy:

-   -   Examples 2 to 4 were synthesized in analogy to Example 1.    -   Example 6 was synthesized in analogy to Example 5.    -   Example 9 to 25, and 27 were synthesized in analogy to Example        26.    -   Example 29 was synthesized in analogy to Example 28.    -   Examples 30 to 61 were synthesized in analogy to Example 8.    -   Example 63 was synthesized in analogy to Example 62.

Example 64 pE-R-P-R-L-C-H-K-G-P-Nle-C-F-OH with a —S—CH₂—C(O)CH₂—S—Linkage Between the Cysteines at Position 6 and 12 [C⁶-C¹²] AnalyticalMethod Method D (HRMS);

Eluent A: water+0.05% Formic acid+3.75 mM ammonium acetate, Eluent B:Acetonitrile+0.04% Formic acid.Gradient: from 2 to 98% B in 4.4 min—Flow 1.0 ml/min. Column: AcquityCSH 1.7 μm 2.1*50 mm 50° C.

Example 8((S)-2-((3S,6R,11R,14S,17S,25aS)-14-((1H-imidazol-5-yl)methyl)-17-(4-aminobutyl)-3-butyl-11-((S)-2-((S)-5-guanidino-2-((S)-1-((S)-5-guanidino-2-((S)-5-oxopyrrolidine-2-carboxamido)pentanoyl)pyrrolidine-2-carboxamido)pentanamido)-4-methylpentanamido)-1,4,12,15,18,21-hexaoxodocosahydro-1H-pyrrolo[2,1-j][1,2,5,8,11,14,17,20]dithiahexaazacyclotricosine-6-carboxamido)-3-phenylpropanoicacid) (12 mg, 6.76 μmol) was dissolved in 50 mM sodium phosphate bufferpH6.5 (1.5 ml), into which was added TCEP HCl((tris(2-carboxyethyl)phosphine) (2.91 mg, 10.13 μmol) at RT. Thisreaction mixture was stirred for 1 h at RT. Into above solution wasadded 1,3-dichloropropan-2-one (4.29 mg, 0.034 mmol) at RT, which wasstirred for 30 min at RT. RP-HPLC eluting 15-60% MeCN/water with 0.1%TFA gave Example 64((S)-2-((3S,6R,14R,17S,20S,28aS)-17-((1H-imidazol-5-yl)methyl)-20-(4-aminobutyl)-3-butyl-14-((S)-2-((S)-5-guanidino-24(S)-1-((S)-5-guanidino-2-((S)-5-oxopyrrolidine-2-carboxamido)pentanoyl)pyrrolidine-2-carboxamido)pentanamido)-4-methylpentanamido)-1,4,10,15,18,21,24-heptaoxohexacosahydropyrrolo[2,1i][1,23,4,7,10,13,16,19]dithiahexaazacyclohexacosine-6-carboxamido)-3-phenylpropanoicacid) (6 mg, 2.93 μmol, 43.4% yield). HRMS (method D) [M+1]; 1590.7911(observed), 1590.7912 (expected). Retension time; 3.08 min.

Example 65 pE-R-P-R-L-C-H-K-G-P-Nle-C-F-OH with a Monosulfide LinkageBetween the 2 Cysteines at Position 6 and 12 [C⁶-C¹²]

The mixture of Example 8(S)-2-((3S,6R,11R,14S,17S,25aS)-14-((1H-imidazol-5-yl)methyl)-17-(4-aminobutyl)-3-butyl-11-((S)-2-((S)-5-guanidino-2-((S)-1-((S)-5-guanidino-2-((S)-5-oxopyrrolidine-2-carboxamido)pentanoyl)pyrrolidine-2-carboxamido)pentanamido)-4-methylpentanamido)-1,4,12,15,18,21-hexaoxodocosahydro-1H-pyrrolo[2,1-j][1,2,5,8,11,14,17,20]dithiahexaazacyclotricosine-6-carboxamido)-3-phenylpropanoicacid TFA salts (30 mg, 0.15 mmol) andN,N,N′,N′,N″,N″-hexamethylphosphinetriamine (12.3 mg, 0.75 mmol) in PBSpH 9.2 buffer (1 mL) was stirred at RT for 3 days. The reaction mixturewas purified by preparative HPLC (Sunfire C18, 0.1% TFA in water/MeCN)twice, and the product fraction was lyophilized to a white powder(Example 65: 4 mg, 13.4%). [M+2H]2+(calc.)=752.88,[M+2H]2+(measured)=752.40, [M+3H]3+(calc.)=502.26,[M+3H]3+(measured)=501.94. HPLC (analytical method C), Tr min=6.93.

Example 66 pE-R-P-R-L-C-H-K-G-P-Nle-C-F-OH with a —S—CH₂—C(═Z)—CH₂—S—Linkage Between the 2 Cysteines at Position 6 and 12 [C⁶-C¹²] and Z is

Analytical Method Method D (HRMS);

Eluent A: water+0.05% Formic acid+3.75 mM ammonium acetate, Eluent B:Acetonitrile+0.04% Formic acid.Gradient: from 2 to 98% B in 4.4 min—Flow 1.0 ml/min. Column: AcquityCSH 1.7 μm 2.1*50 mm 50° C.

Into a solution of Example 64((S)-2-((3S,6R,14R,17S,20S,28aS)-17-((1H-imidazol-5-yl)methyl)-20-(4-aminobutyl)-3-butyl-14-((S)-2-((S)-5-guanidino-24(S)-1-((S)-5-guanidino-2-((S)-5-oxopyrrolidine-2-carboxamido)pentanoyl)pyrrolidine-2-carboxamido)pentanamido)-4-methylpentanamido)-1,4,10,15,18,21,24-heptaoxohexacosahydropyrrolo[2,1-i][1,23,4,7,10,13,16,19]dithiahexaazacyclohexacosine-6-carboxamido)-3-phenylpropanoicacid) (11.5 mg, 5.62 μmol) and(S)-1-(aminooxy)-19-carboxy-2,7,16,21-tetraoxo-9,12-dioxa-3,6,15,20-tetraazaoctatriacontan-38-oicacid compound with 2,2,2-trifluoroacetic acid (1:1) (9.19 mg, 0.011mmol) in 100 nM Na phosphate buffer pH6.0 (1 ml) was added aniline(2.051 μl, 0.022 mmol) at RT. Addition of DMSO (50 μl) gave homogeneoussolution. This reaction mixture was stirred at RT for 2 h. RP-HPLCeluting 15-60% MeCN/water with 0.1% TFA gave Example 66(1-((Z)-((3S,6R,14R,17S,20S,28aS)-17-((1H-imidazol-5-yl)methyl)-20-(4-aminobutyl)-3-butyl-6-((S)-1-carboxy-2-phenylethylcarbannoyl)-14-((S)-2-((S)-5-guanidino-2-((S)-1-((S)-5-guanidino-2-((S)-5-oxopyrrolidine-2-carboxamido)pentanoyl)pyrrolidine-2-carboxamido)pentanamido)-4-methylpentanamido)-1,4,15,18,21,24-hexaoxodocosahydropyrrolo[2,1-i][1,23,4,7,10,13,16,19]dithiahexaazacyclohexacosin-10(1H,9H,11H)-ylidene)aminooxy)-19-carboxy-2,7,16,21-tetraoxo-9,12-dioxa-3,6,15,20-tetraazaoctatriacontan-38-oicacid) (4.5 mg, 1.646 μmol, 29.3% yield). HRMS (method D) [(M+3)/3];759.7487 (observed), 759.7462 (expected). Retension time; 4.12 min.

Synthesis of the(S)-1-(aminooxy)-19-carboxy-2,7,16,21-tetraoxo-9,12-dioxa-3,6,15,20-tetraazaoctatriacontan-38-oicacid compound with 2,2,2-trifluoroacetic acid (1:1)

1-Chlorotrytyl chloride resin (1.55 mmol/g) (0.500 g, 0.775 mmol) in 100mL glassware was swollen in DCM (20 ml) for 30 min and it was drained. asuspension of 2-(aminooxy)acetic acid hemihydrochloride (0.338 g, 3.10mmol) and DIPEA (1.354 ml, 7.75 mmol) in NMP (7 ml)/DCM (4 ml) was addedto the resin, which was shaken for 5 h. Solvent was drained. resin wasrinsed with DCM/MeOH/DIPEA (17/2/1, 40 mL), DCM(50 mL), NMP(50 mL)DCM(50 mL) respectively. resulting resin was dried with KOH/NaOHovernight.

Resin (0.775 mmol) in 100 mL glassware was swollen in DCM (20 ml) for 30min and it was drained. Into a suspension of (9H-fluoren-9-yl)methyl2-aminoethylcarbamate hydrochloride (0.081 g, 0.775 mmol), HOAt (0.422g, 3.10 mmol) and DIPEA (1.354 ml, 7.75 mmol) in NMP (8 ml) was addedHBTU (1.176 g, 3.10 mmol) in NMP (2.5 ml), which was shaken for 2 h atRT. solvent was drained. resin was rinsed with NMP(10 mL), DCM(10 mL)respectively. The resulting resin was dried overnight.

Resin (0.775 mmol) was charged into reaction vassel. 10 mL of 20%PIPERIDINE/NMP was added into resin, which suspension was agitated at RTfor 5 min. After solvent was drained, additional 10 mL of 20%PIPERIDINE/NMP was added and agitated for 20 min at RT. Solution of HOAt(0.316 g, 2.325 mmol) and1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azadodecan-12-oic acid (0.896g, 2.325 mmol) in NMP (8 ml) was added into resin and DIC (0.362 ml,2.325 mmol) in NMP (1 ml) was added. reaction mixture was agitated for 2h at RT. Resin was filtered off and rinsed with NMP (10 ml) four times.The resulting resin was dried overnight.

Resin (0.775 mmol) was charged into reaction vassel. 10 mL of 20%PIPERIDINE/NMP was added into resin, which suspension was agitated at RTfor 5 min. After solvent was drained, additional 10 mL of 20%PIPERIDINE/NMP was added and agitated for 20 min at RT. Solution of HOAt(0.316 g, 2.325 mmol) and Fmoc-Glu-OtBu (0.989 g, 2.325 mmol) in NMP (8ml) was added into resin and DIC (0.362 ml, 2.325 mmol) in NMP (2.00 ml)was added. The reaction mixture was agitated for 2 h at RT. Resin wasfiltered off and rinsed with NMP (10 ml) four times. The resulting resinwas dried overnight.

Resin (0.775 mmol) was charged into reaction vassel. 10 mL of 20%PIPERIDINE/NMP (0.775 mmol) was added into resin, which suspension wasagitated at RT for 5 min. after solvent was drained, additional 10 mL of20% PIPERIDINE/NMP (0.775 mmol) (0.775 mmol) was added and agitated for20 min at RT. Solution of 18-tert-butoxy-18-oxooctadecanoic acid (0.862g, 2.325 mmol) and HOAt (0.316 g, 2.325 mmol) in NMP (8 ml) was addedinto resin and DIC (0.362 ml, 2.325 mmol) in NMP (2.00 ml) was added.The reaction mixture was agitated for 4 h at RT. Resin was filtered offand rinsed with NMP (10 ml) four times. The resulting resin was driedovernight.

Resin (0.775 mmol) was treated with 20 mL of cleavage cocktail(TFA/TIPS/water=95/2.5/2.5) for 1.5 h at RT. Resin was removed byfiltration and rinsed with TFA. The filtrate was concentrated in vacuo.RP-HPLC with C18 column eluting 15-50% MeCN/water with 0.1% TFA gave(S)-1-(aminooxy)-19-carboxy-2,7,16,21-tetraoxo-9,12-dioxa-3,6,15,20-tetraazaoctatriacontan-38-oicacid with 2,2,2-trifluoroacetic acid (1:1) (207 mg, 0.294 mmol, 37.9%yield). HRMS (method D) [M+1]; 704.4459 (observed), 704.4486 (expected).Retension time; 2.63 min.

The polypeptides of Examples 1-66 can be purified and isolated asdescribed supra and/or by a combination of conventional purificationtechniques such as solvent extraction, column chromatography, liquidchromatography and recrystallization. Where the polypeptide isolated inthe above Examples is a free compound, it can be converted to a suitablesalt by the known method. Therefore, Peptides of Examples 1-66 can beconverted into their corresponding salt (e.g. hydrochloride,hydrobromide, sulfate, phosphate, citrate, acetate, lactate or anotherpharmaceutical salt for suitable for injection) with a polypeptide:saltratio ranging from 1:1 to 1:4. For example, polypeptides of Examples1-66 can be dissolved in water and converted into a salt usingion-exchange resins. Conversely where the isolated peptide is a salt, itcan be converted to the free peptide by known method or directly to adifferent salt with the aid of ion-exchange resins.

The polypeptide in the examples below have been found to have EC₅₀values in the range of about 0.01 nM to about 1100 nM for APJ receptorpotency. The polypeptides in the examples below have been found to havea plasma stability higher than 2 minutes, higher than 5 minutes, higherthan 10 minutes, higher than 20 minutes, higher than 50 minutes andhigher than 60 minutes.

It can be seen that the polypeptides of the invention are useful asagonist of the APJ receptor and therefore useful in the treatment ofdiseases and conditions responsive the activation of the APJ receptor,such as the diseases disclosed herein.

Having thus described exemplary embodiments of the present invention, itshould be noted by those of ordinary skill in the art that the withindisclosures are exemplary only and that various other alternatives,adaptations, and modifications may be made within the scope of thepresent invention. Accordingly, the present invention is not limited tothe specific embodiments as illustrated therein.

What is claimed is: 1-7. (canceled)
 8. A polypeptide having Formula VI:

or an amide, an ester or a salt of the polypeptide wherein: X1 is theN-terminus of the polypeptide and is either absent or is selected frompE, R, Isn, Q, A, K, and 5-amino-valeric acid; X2 is R, A, r, N-Me-R, K,H, hF, hK, F, E or Orn; X4 is R, A, r, N-Me-R, F, E; X5 is L, Cha, A,D-L, N-Me-L, K, D, 4-PhF or F; X8 is K, k, F, f, A, hF, N-Me-R, E or4-amino-Isn; X9 is G, N-Me-G, A, D, L, R or Aib; X10 is P, A, p, 4-PhPor pipecolic acid, X11 is M, D-Nle, Nle, N-Me-Nle, M(O), A, F, Y, L, K,3-PyA or Cha; and X13 is the C-terminus and is absent or is selectedfrom F, f, N-Me-F, Nal, D-Nal, 3-Br—F, (S)-β-3-F, I, A, a, K, Dap, H andE; wherein: Nle is L-norleucine; hF is L-homophenylalanine; hK isL-lysine; Nal is L-naphathaline; Orn is ornithine; Aib isα-aminoisobutyric acid; Dab is (S)-diaminobutyric acid; Dap is(S)-2,3-diaminopropionic acid; M(O) is methionine sulfone; Cha is(S)-β-cyclohexylalanine; 4-amino-Isn is 4-aminopiperidine-4-carboxylicacid; Isn is isonipecotinoyl; pE is L-pyroglutamic acid; 3-PyA is3-(3-pyridyl)-L-alanine; 4-PhF is 4-Phenyl-L-phenylalanine; wherein theN-terminus and the C-terminus optionally form a ring together with 1, 2,3 or 4 glycine amino acids; and wherein the amino group in the sidechain of K, Orn, Dab, Dap, hK or 4-amino-Isn is optionally linked to alipophilic group via an amide bond. 9-12. (canceled)
 13. The polypeptideaccording to claim 8 wherein X1 is pE; or an amide, an ester or a saltof the polypeptide.
 14. The polypeptide according to 8 wherein X1 isabsent; or an amide, an ester or a salt of the polypeptide.
 15. Thepolypeptide according to claim 14 wherein the N-terminus is an amide; ora salt of the polypeptide.
 16. The polypeptide according to claim 15wherein the N-terminus is an amide of Formula —NHR and R is Acetyl,benzoyl, phenacyl, succinyl, octanoyl, 4-phenylbutanoyl,4-Cl-Ph-(CH₂)₃C(O)—, or Ph-CH₂CH₂NHC(O)—; or a salt of the polypeptide.17. The polypeptide of claim 8 wherein the N-terminus is an amide ofFormula NHR1 wherein R1 is CH₃C(O)—, CH₃—(O—CH₂CH₂)_(m)—C(O)—,Palmitoyl(O2Oc)_(p), Myristoyl(O2Oc)_(p), Lauroyl(O2Oc)_(p) orPh-CH₂CH₂NHC(O)—; and wherein p is an integer of 1 to 4; m is an integerof 1 to 12; Lauroyl(O2Oc) is C₁₁H₂₃C(O)NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)—;Myristoyl(O2Oc) is C₁₃H₂₇C(O)NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)—;Palmitoyl(O2Oc) is C₁₅H₃₁C(O)NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)—; or a saltof the polypeptide
 18. The polypeptide according to claim 8 wherein X13is F; or an amide, an ester or a salt of the polypeptide.
 19. Thepolypeptide according to claim 8 wherein X13 is absent; or an amide, anester or a salt of the polypeptide.
 20. The polypeptide according toclaim 19 wherein the C-terminus is an amide; or a salt of thepolypeptide.
 21. The polypeptide according to claim 20 wherein theC-terminus is an amide of Formula —C(O)—R2 and R2 is —NH₂, —NH-Me,—NH—NHBn, or —NH—(CH₂)₂-Ph; or a salt of the polypeptide.
 22. Thepolypeptide according to claim 8 wherein X5 is L; or an amide, an esteror a salt of the polypeptide.
 23. (canceled)
 24. The polypeptideaccording to claim 8 wherein X8 is K; or an amide, an ester or a salt ofthe polypeptide.
 25. The polypeptide according to claim 8 wherein X9 isG; or an amide, an ester or a salt of the polypeptide.
 26. Thepolypeptide according to claim 8 wherein X11 is Nle, or an amide, anester or a salt of the polypeptide.
 27. The polypeptide according toclaim 8: wherein the two amino acids labeled with “*” represent theamino acids forming a disulfide and wherein the two amino acids labeledwith “**” represent the amino acids forming a disulfide via their sidechain or an amide, an ester or a salt of the polypeptide.
 28. (canceled)29. A method of treating or preventing a disease or disorder responsiveto the agonism of the APJ receptor, in a subject in need thereof,comprising administering to the subject a therapeutically effectiveamount of a polypeptide according to claim 8 or an amide, an ester or asalt thereof.
 30. The method of claim 29 wherein the disease or disorderis selected from acute decompensated heart failure (ADHF), chronic heartfailure, pulmonary hypertension, atrial fibrillation, Brugada syndrome,ventricular tachycardia, atherosclerosis, hypertension, restenosis,ischemic cardiovascular diseases, cardiomyopathy, cardiac fibrosis,arrhythmia, water retention, diabetes (including gestational diabetes),obesity, peripheral arterial disease, cerebrovascular accidents,transient ischemic attacks, traumatic brain injuries, amyotrophiclateral sclerosis, burn injuries (including sunburn) and preeclampsia.31-35. (canceled)
 36. A pharmaceutical composition comprising atherapeutically effective amount of a polypeptide according to claim 8or an amide, an ester or a salt thereof, and one or morepharmaceutically acceptable carriers.